Macrocyclic ligands with picolinate group(s), complexes thereof and also medical uses thereof

ABSTRACT

A macrocycle compound of general formula (X) 
                         
in which Y 1  represents a C(O)OH group or a group of formula (II)

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.16/065,661, filed on Jun. 22, 2018, which is a U.S. National Phaseapplication of PCT/EP2016/082644, filed on Dec. 23, 2016, claiming thebenefit of French Application No. 1563346, filed on Dec. 24, 2015, eachof which is incorporated herein by reference in its entirety.

The present invention relates to novel macrocyclic ligands and alsocomplexes thereof, especially radioactive complexes, and the usesthereof in medical imaging and/or in therapy, especially ininterventional radiology.

The present invention also relates to a novel process for preparingligands such as according to the invention, and also the preparationintermediates thereof.

The need for targeted and personalized treatments in oncology has led tothe development of novel therapeutic strategies based on early detectiontools combined with more specific and more efficient vectorizedtreatments.

Interventional radiology is a very promising direction in individualizedmedicine. It allows combination in the same sequence of precisediagnosis of the lesion or tumor and/or instantaneous treatment thereof,guided and controlled by images. It is described as minimally invasivesurgery and can as a result be performed as outpatient treatment, whichallows saving of many expensive days of hospitalization for an efficacythat is often comparable with that of conventional surgery.Interventional radiology may thus represent an alternative or acomplement to conventional surgical treatment.

Interventional radiology allows access to a lesion or tumor locatedinside the body to perform a diagnostic action (for example sampling) ora therapeutic action. Imaging by fluoroscopy, echography, scanner or MRIallows pinpointing, guiding and optimum control of the medical gesture.

There is thus a need for novel molecules that can be used in medicalimaging and/or in therapy, in particular in interventional radiology.More particularly, there is a need for ligands which can complexchemical elements, in particular metals, so as to obtain complexes thatcan be used in medical imaging and/or in therapy, in particular ininterventional radiology.

Such ligands must especially be stable and must complex metals stronglyenough for them to reach their target without diffusing into othersensitive organs or tissues such as bone, the lungs and the kidneys.

The aim of the present invention is to provide novel ligands forcomplexing chemical elements, in particular radioelements.

The aim of the present invention is also to provide novel complexes, inparticular radioactive complexes.

The aim of the present invention is to provide ligands and/or complexesthat are particularly useful in medical imaging and/or in therapy,especially in cancer treatment.

The aim of the present invention is also to provide a pharmaceuticalcomposition comprising complexes which allow the medical imaging,targeting and/or treatment of cancers.

The aim of the present invention is to provide a novel process forpreparing these ligands.

The present invention relates to a compound of general formula (I)below:

in which:

-   -   R₁, R₂, R₃, R₄, R₅ and R₆ represent, independently of each        other, H, a (C₁-C₂₀)alkyl group or a        (C₁-C₂₀)alkylene-(C₆-C₁₀)aryl group;

said alkyl, alkylene and aryl groups possibly being substituted with oneor more substituents chosen from organic acid functions, preferably fromthe group constituted by —COOH, —SO₂OH, —P(O)(OH)₂ and —O—P(O)(OH)₂;

-   -   X₁, X₂ and X₃ are chosen, independently of each other, from the        group constituted by: H, —C(O)N(Re)(Rd), (C₁-C₂₀)alkyl,        (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl and (C₆-C₁₀)aryl, with Re and        Rd being, independently of each other, H or a (C₁-C₂₀)alkyl        group,

said alkyl, alkenyl and alkynyl groups possibly comprising one or moreheteroatoms and/or one or more (C₆-C₁₀)arylenes in their chain andpossibly being substituted with a (C₆-C₁₀)aryl;

said alkyl, alkenyl, alkynyl and aryl groups possibly being substitutedwith one or more substituents chosen from organic acid functions,preferably from the group constituted by —COOH, —SO₂OH, —P(O)(OH)₂ and—O—P(O)(OH)₂;

-   -   Y₁, Y₂ and Y₃ represent, independently of each other, a C(O)OH        group or a group of formula (II) below:

in which:

the radicals Ri are chosen, independently of each other, from the groupconstituted by:

H, halogen, N₃, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl and(C₆-C₁₀)aryl, said alkyl, alkenyl and alkynyl groups possibly comprisingone or more heteroatoms and/or one or more (C₆-C₁₀)arylenes in theirchain and possibly being substituted with a (C₆-C₁₀)aryl;

said alkyl, alkenyl, alkynyl and aryl groups possibly being substitutedwith one or more substituents chosen from organic acid functions,preferably from the group constituted by —COOH, —SO₂OH, —P(O)(OH)₂ and—O—P(O)(OH)₂;

and

at least one of the radicals Y₁, Y₂ and Y₃ being a group of formula(II);

or a pharmaceutically acceptable salt thereof.

-   -   According to one embodiment, the present invention relates to a        compound of general formula (I) below:

in which:

-   -   R₁, R₂, R₃, R₄, R₅ and Re represent, independently of each        other, H, a (C₁-C₂₀)alkyl group or a        (C₁-C₂₀)alkylene-(C₆-C₁₀)aryl group;    -   X₁, X₂ and X₃ are chosen, independently of each other, from the        group constituted by: H, —C(O)N(Re)(Rd), (C₁-C₂₀)alkyl,        (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl and (C₆-C₁₀)aryl, with Re and        Rd being, independently of each other, H or a (C₁-C₂₀)alkyl        group,    -   said alkyl, alkenyl and alkynyl groups possibly comprising one        or more heteroatoms and/or one or more (C₆-C₁₀)arylenes in their        chain and possibly being substituted with a (C₆-C₁₀)aryl;    -   Y₁, Y₂ and Y₃ represent, independently of each other, a C(O)OH        group or a group of formula (II) below:

in which:

-   -   the radicals Ri are chosen, independently of each other, from        the group constituted by: H, halogen, N₃, (C₁-C₂₀)alkyl,        (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl and (C₆-C₁₀)aryl,    -   said alkyl, alkenyl and alkynyl groups possibly comprising one        or more heteroatoms and/or one or more (C₆-C₁₀)arylenes in their        chain and possibly being substituted with a (C₆-C₁₀)aryl; and        at least one of the radicals Y₁, Y₂ and Y₃ being a group of        formula (II);        or a pharmaceutically acceptable salt thereof.

The inventors have developed novel ligand-metal complexes (complexesalso known as chelates) from the pyclene macrocycle(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene), variouslysubstituted with acetate and/or picolinate groups(6-methylene-2-pyridinecarboxylic acid). The pyclene macrocycle has thefollowing formula:

Surprisingly, the complexes according to the invention have goodthermodynamic stability and also good kinetic inertia. Furthermore,equally surprisingly, the inventors have discovered that the complexesaccording to the invention can be dissolved in an iodinated oil such asLipiodol®, which is an iodinated oil manufactured and sold by thecompany Guerbet and which is constituted by ethyl esters of iodinatedfatty acids of poppy oil. Thus, the complexes according to the inventiondissolved in an iodinated oil such as Lipiodol® can be vectorizedespecially toward the liver and can allow the visualization and/ortreatment of cancers, for example liver cancer.

These complexes also have a good radiochemical yield for extraction intoan iodinated oil such as Lipiodol®. They in particular show goodincorporation of radioactivity into an iodinated oil such as Lipiodol®and good stability of the radioactive Lipiodol® solution in in vitrotests.

In particular, the combination of the properties of Lipiodol®vectorization, of therapeutic efficacy of the radioelements and the goodtolerability of these products make it possible to propose a therapeuticcancer treatment that is safe and easier to perform.

Vectorization of the complexes according to the invention with aniodinated oil such as Lipiodol® makes it possible especially to avoidpoor delivery of the complexes while reducing the risk of adverseeffects in healthy organs, in particular healthy liver or in theextra-hepatic organs, and makes it possible to achieve the effectiveradioactivity dose in the tumor.

More particularly, this vectorization facilitates the work of theinterventional radiologist at the time of injection of the complexesaccording to the invention. For example, during intra-arterial injectionmonitored by fluoroscopy, the radiologist's gesture will be more preciseand safer, allowing adjustment of the rate of delivery of the complexesas a function of the uptake by the tumor of the complexes according tothe invention.

DEFINITIONS

The term “ligand” means a compound that is capable of complexing achemical element such as a metal, preferably a radioelement. Accordingto one embodiment, the ligands within the meaning of the invention arein anionic form and can complex radioelements in cationic form, forexample metal cations in oxidation state (III). According to the presentinvention, the compounds of formula (I) are ligands.

The term “radioelement” means any known radioisotope of a chemicalelement, whether it is natural or artificially produced. According toone embodiment, the radioelement is chosen from yttrium and lanthanideradioisotopes. The term “lanthanides” denotes atoms chosen from thegroup constituted by: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er,Tm, Yb and Lu.

The term “complex” means the combination of a ligand as defined abovewith a chemical element, preferably a radioelement as defined above. Theterm “complex” is synonymous with “chelate”.

The “thermodynamic stability” represents the affinity of the ligand fora given element, in particular a given metal. It is the equilibriumconstant for the complexation reaction:

-   -   Metal+Ligand        Complex        the mathematical expression of which is as follows:

$K = \frac{\lbrack{Metal}\rbrack \times \lbrack{Ligand}\rbrack}{\lbrack{Complex}\rbrack}$

The values are generally expressed in decimal logarithm form log K.According to one embodiment, the complexes according to the inventionhave strong affinity. According to one embodiment, the complexesaccording to the invention have an equilibrium thermodynamic constant atleast equal to 16 (log K at least equal to 16).

The complexes formed according to the equilibrium reaction describedabove are capable of dissociating under the action of various factors(pH, presence of metals or competing ligands). This dissociation mayhave substantial consequences in the context of using the complexes inhuman medicine, since it entails release of the metal into the body. Inorder to limit this risk, slow dissociation complexes are desired, i.e.complexes with good kinetic inertia. The kinetic inertia may bedetermined via dissociation tests in acidic medium. These experimentslead to the determination for each complex of a half-life time (T_(1/2))under defined conditions.

In the context of the invention, the term “treating”, “treatment” or“therapeutic treatment” means reversing, relieving or inhibiting theprogress of the disorder or complaint to which this term is applicable,or one or more symptoms of such a disorder.

The term “medical imaging” denotes means for the acquisition andrestitution of images of the human or animal body by means of variousphysical phenomena such as x-ray absorption, nuclear magnetic resonance,ultrasound wave reflection or radioactivity. According to oneembodiment, the term “medical imaging” refers to x-ray imaging, MRI(magnetic resonance imaging), single-photon emission tomography (SPECT:single-photon emission computed tomography), positron emissiontomoscintigraphy (PET) and luminescence. Preferably, the medical imagingmethod is x-ray imaging. According to a particular embodiment, themedical imaging method is MRI if the complex according to the inventioncomprises Gd(III), SPECT if the complex according to the inventioncomprises a gamma emitter and PET if the complex according to theinvention comprises a beta+ emitter.

The capacity of contrast agents to accelerate the rates of relaxation1/T1 and 1/T2 of the protons of water is measured by means of amagnitude known as relaxivity. The relaxivity (r) of a contrast agent isespecially defined as the rate of relaxation, normalized by theconcentration of the contrast agent.

The term “(C₁-C₂₀)alkyl” denotes saturated aliphatic hydrocarbons, whichmay be linear or branched and comprise from 1 to 20 carbon atoms.Preferably, the alkyls comprise from 1 to 15 carbon atoms, for example6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms. The term “branched”means that an alkyl group is substituted on the main alkyl chain.

The term “(C₁-C₂₀)alkylene” denotes an alkyl radical as defined above,which is divalent.

The term “(C₂-C₂₀)alkene” denotes an alkyl as defined above, comprisingat least one carbon-carbon double bond.

The term “(C₂-C₂₀)alkyne” denotes an alkyl as defined above, comprisingat least one carbon-carbon triple bond.

The term “(C₆-C₁₀)aryl” denotes monocyclic, bicyclic or tricyclichydrocarbon-based aromatic compounds, in particular phenyl and naphthyl.

The term “arylene” denotes an aryl as defined above, which is divalent,in particular phenylene and naphthylene.

According to one embodiment, the term “halogen” denotes F, Cl, Br, I andAt.

Among the heteroatoms, mention may be made especially of P, N, O and S,preferably N and O. According to a particular embodiment, the compoundsof general formula (I) comprise 1 or 2 heteroatoms. Preferably,—O—(C₁-C₂₀)alkyl (also known as alkoxy groups), —O—(C₂₋₂₀)alkenyl and—O—(C₂₋₂₀)alkynyl groups are present.

The term “Lipiodol” refers to an iodinated oil and preferentially to thepharmaceutical specialty Lipiodol®, which is an injectable solutionmanufactured and sold by Guerbet and constituted by ethyl esters ofiodinated fatty acids of poppy oil. Lipiodol® is a product that is usedespecially for visualization, localization and/or vectorization in thecourse of transarterial chemoembolization of hepatocellular carcinoma atthe intermediate stage in adults, and also for diagnosis via theselective hepatic arterial route of the hepatic extension of malignantor non-malignant hepatic lesions.

The term “organic acid” (or organic acid function) means an organiccompound (or an organic function) which has acidic properties, i.e.which is capable of releasing an H⁺ or H₃O⁺ cation in aqueous media.Among the organic acids, mention may be made of carboxylic acids,sulfonic acids, phosphates and phosphonates. The organic acid functionsaccording to the invention are preferably chosen from the groupconstituted by —COOH, —SO₂OH, —P(O)OH, —P(O)(OH)₂ and —O—P(O)(OH)₂, andmore preferentially —COOH. Such acid functions are salifiable and may bein the basic form thereof. In particular, these acid functions are inthe form of pharmaceutically acceptable salts, as defined below; forexample, in the form of the sodium or meglumine(1-deoxy-1-(methylamino)-D-glucitol or N-methyl-D-glucamine) salt.

The Iodinated Oils

The term “fatty acid” denotes saturated or unsaturated aliphaticcarboxylic acids bearing a carbon chain of at least 4 carbon atoms.Natural fatty acids bear a carbon chain of 4 to 28 carbon atoms(generally an even number). The term “long-chain fatty acid” refers to alength of 14 to 22 carbons, and “very-long-chain fatty acid” refers tomore than 22 carbons. Conversely, the term “short-chain fatty acid”refers to a length of 4 to 10 carbons, especially 6 to 10 carbon atoms,in particular 8 or 10 carbon atoms. A person skilled in the art knowsthe associated nomenclature and in particular uses:

-   -   Ci-Cp to denote a fatty acid range from Ci to Cp    -   Ci+Cp, the total of the Ci fatty acids and of the Cp fatty acids        For example:    -   fatty acids of 14 to 18 carbon atoms is written as “C₁₄-C₁₈        fatty acids”    -   the total of the C16 fatty acids and of the C18 fatty acids is        written as C16+C18    -   for a saturated fatty acid, a person skilled in the art will use        the following nomenclature Ci: 0, in which i is the number of        carbon atoms in the fatty acid. Palmitic acid, for example, will        be denoted by the nomenclature (C16:0).    -   for an unsaturated fatty acid, a person skilled in the art will        use the following nomenclature Ci; x n-N in which N will be the        position of the double bond in the unsaturated fatty acid        starting from the carbon opposite the acid group, i is the        number of carbon atoms in the fatty acid and x is the number of        double bonds (unsaturations) in this fatty acid. Oleic acid, for        example, will be denoted by the nomenclature (C18;1 n-9).

Advantageously, the iodinated oil according to the invention comprisesor is constituted by iodinated fatty acid derivatives, preferentiallyethyl esters of iodinated fatty acids, more preferentially ethyl estersof iodinated fatty acids of poppy oil, of olive oil, of rapeseed oil, ofgroundnut oil, of soybean oil or of walnut oil, and even morepreferentially ethyl esters of iodinated fatty acids of poppy oil or ofolive oil. More preferentially, the iodinated oil according to theinvention comprises or is constituted by ethyl esters of iodinated fattyacids of poppy (also known as black poppy or Papaver somniferum var.nigrum) oil. Poppy oil, also known as poppy seed oil, preferentiallycontains more than 80% of unsaturated fatty acids (in particularlinoleic acid (C18:2 n-6) and oleic acid (C18:1 n-9)) including at least70% of linoleic acid and at least 10% of oleic acid. The iodinated oilis obtained from the total iodination of an oil such as poppy oil underconditions allowing one iodine atom bond for each double bond of theunsaturated fatty acids (Wolff et al. 2001, Medicine 80, 20-36) followedby transesterification.

The iodinated oil according to the invention preferentially containsfrom 29% to 53% (m/m) and more preferentially 37% to 39% (m/m) ofiodine.

As examples of iodinated oils, mention may be made of Lipiodol®,Brassiodol® (derived from rapeseed (Brassica compestis) oil), Yodiol®(derived from groundnut oil), Oriodol® (derived from poppy oil in fattyacid triglyceride form) and Duroliopaque® (derived from olive oil).

Preferentially, the iodinated oil is Lipiodol®, which is an iodinatedoil used as a contrast product and in certain interventional radiologyprocedures. This oil is a mixture of ethyl esters of iodinated andnon-iodinated fatty acids of poppy seed oil. It consists predominantly(in particular more than 84%) of a mixture of ethyl esters of iodinatedlong-chain fatty acids (in particular C18 fatty acids) derived frompoppy seed oil, preferentially as a mixture of ethyl monoiodostearateand of ethyl diiodostearate. The iodinated oil may also be an oil basedon the ethyl ester of monoiodostearic acid (C18:(0) derived from oliveoil. A product of this type, known as Duroliopaque®, was marketed a fewyears ago.

The main characteristics of Lipiodol® are the following:

Compounds Proportions in the fatty acid mixture Ethyl palmitate 4.6 to6.7% (m/m), preferentially 4.8% (m/m) (ethyl C16:0) Ethyl stearate 0.8to 1.9% (m/m), preferentially 1.2% (m/m) (ethyl C18:0) Ethyl 11.3 to15.3% (m/m), preferentially 13.4% (m/m) monoiodostearate Ethyl 73.5 to82.8% (m/m), preferentially 78.5% (m/m) diiodostearate Othercharacteristics of Lipiodol ®: Iodine 37% to 39% (m/m) (i.e. 480 mg/ml)Viscosity at 37° C. 25 mPa · s at 20° C. 50 mPa · s Density 1.268-1.290g/cm³ at 20° C., preferentially 1.28Compounds of General Formula (I)

The compounds of general formula (I) may have chiral centers and may bein racemic or enantiomeric form. The compounds of general formula (I)are included in their various forms: diastereoisomers, enantiomers orracemic mixture.

According to one embodiment, the compounds of general formula (I) are insalt form, preferably in the form of a pharmaceutically acceptable salt.

The term “pharmaceutically acceptable salt” especially denotes saltswhich allow the properties and the biological efficacy of the compoundsaccording to the invention to be conserved. Examples of pharmaceuticallyacceptable salts are found in Berge et al., ((1977) J. Pharm. Sd, vol.66, 1). For example, the compounds of general formula (I) are in theform of the sodium or meglumine (1-deoxy-1-(methylamino)-D-glucitol orN-methyl-D-glucamine) salt.

The invention also relates to the optical isomers (enantiomers),geometrical isomers (cis/trans or Z/E), tautomers and solvates such ashydrates of the compounds of formula (I).

According to one embodiment, X₁, X₂ and X₃ are chosen, independently ofeach other, from the group constituted by: H, (C₁-C₂₀)alkyl,(C₂-C₂₀)alkenyl and (C₂-C₂₀)alkynyl, said alkyl, alkenyl and alkynylgroups possibly comprising one or more heteroatoms in their chain.

According to a particular embodiment, X₁, X₂ and X₃ are chosen,independently of each other, from the group constituted by: H and(C₁-C₂₀)alkyl. More particularly, X₁, X₂ and X₃ are H.

According to one embodiment, in the compounds of general formula (I),when the radicals Y₁, Y₂ or Y₃ represent a group of formula (II), thecorresponding radicals R₁ and R₂, R₃ and R₄ or R₅ and R₆ represent H.

According to one embodiment, the radicals R₁, R₂, R₃, R₄, R₅ and R₆represent, independently of each other, H or a (C₁-C₂₀)alkyl group.According to a particular embodiment, R₁, R₂, R₃, R₄, R₅ and R₆represent H.

According to one embodiment, the radicals Ri are chosen, independentlyof each other, from the group constituted by: H, (C₁-C₂₀)alkyl,(C₂-C₂₀)alkenyl and (C₂-C₂₀)alkynyl, said alkyl, alkenyl and alkynylgroups possibly comprising one or more heteroatoms chosen from N, O andS.

According to one embodiment, the radicals Ri are chosen, independentlyof each other, from the group constituted by: H, (C₁-C₂₀)alkyl,(C₂-C₂₀)alkenyl and (C₂-C₂₀)alkynyl.

According to a particular embodiment, the radicals Ri are chosen,independently of each other, from the group constituted by: H and(C₂-C₁₅)alkynyl.

According to one embodiment, the group of formula (II) is the followinggroup:

According to a particular embodiment:

-   -   R₁, R₂, R₃, R₄, R₅ and R₆ represent H; and    -   the radicals Y₁, Y₂ and Y₃ represent either a C(O)OH group or a        compound having the following formula:

a represents the bond to the carbon atom of the group C(R₁)(R₂),C(R₃)(R₄) or C(R₅)(R₆).

According to one embodiment, the ligand according to the invention hasthe general formula (I-1) below:

in which:

-   -   R₁, R₂, R₃, R₄, R₅ and R₆ represent, independently of each        other, H or a (C₁-C₂₀)alkyl group;    -   Y₁, Y₂ and Y₃ represent, independently of each other, a C(O)OH        group or a group of formula (II) below:

in which:

-   -   the radicals Ri are chosen, independently of each other, from        the group constituted by: H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl and        (C₂-C₂₀)alkynyl,    -   said alkyl, alkenyl and alkynyl groups possibly comprising one        or more heteroatoms in their chain; and        at least one of the radicals Y₁, Y₂ and Y₃ being a group of        formula (II);        or a pharmaceutically acceptable salt thereof.

According to one embodiment, the compounds of general formula (I) may besymmetrical or dissymmetrical. The compounds of general formula (I) aresymmetrical when the groups —C(R₁)(R₂)—Y₁ and —C(R₅)(R₆)—Y₃ areidentical. The compounds of general formula (I) are dissymmetrical whenthe groups —C(R₁)(R₂)—Y₁ and —C(R₅)(R₆)—Y₃ are different. According toone embodiment, the alkyl, alkenyl, alkynyl and aryl groups present inthe radicals Ri are optionally substituted with one or moresubstituents, preferably one substituent, chosen from the groupconstituted by —COOH, —SO₂OH, —P(O)(OH)₂ and —O—P(O)(OH)₂; whereas thealkyl, alkenyl, alkynyl, alkylene and aryl groups of the radicals R₁ toR₆ and X₁ to X₃ are not substituted with said groups.

According to one embodiment, in the radicals X₁, X₂ and X₃, said alkyl,alkenyl, alkynyl and aryl groups, preferably alkyl groups, may beoptionally substituted with one or more substituents, preferably onesubstituent, chosen from the group constituted by —COOH, —SO₂OH,—P(O)(OH)₂ and —O—P(O)(OH)₂.

According to one embodiment, in the radicals R₁, R₂, R₃, R₄, R₅ and R₆,the alkyl, alkylene and aryl groups, preferably alkyl groups, may beoptionally substituted with one or more substituents, preferably onesubstituent, chosen from the group constituted by —COOH, —SO₂OH,—P(O)(OH)₂ and —O—P(O)(OH)₂.

According to one embodiment, in the radicals Ri, the alkyl, alkenyl,alkynyl and aryl groups, preferably alkynyl groups, may be optionallysubstituted with one or more substituents, preferably one substituent,chosen from the group constituted by —COOH, —SO₂OH, —P(O)(OH)₂ and—O—P(O)(OH)₂.

Preferably, said substituent is a —COOH group.

According to one embodiment, the compound of formula (I) is chosen fromthe group constituted by the following compounds:

or a pharmaceutically acceptable salt thereof.

According to a particular embodiment, the compound of formula (I) is thefollowing compound:

or a pharmaceutically acceptable salt thereof.Complexes

The invention also relates to a complex of a compound of formula (I) ora salt thereof, as defined above, with a chemical element M, preferablya metal.

According to one embodiment, the chemical element M is a metal cationchosen from the group constituted by bismuth(III), lead(II), copper(II),copper(I), gallium(III), zirconium(IV), technetium(III), indium(III),rhenium(VI), astatine(III), yttrium(III), samarium(III), actinium(III),lutetium(III), terbium(III), holmium(III), gadolinium(III),europium(III) and yttrium (III), preferably gadolinium(III).

According to a particular embodiment, the chemical element M is aradioelement chosen from the group constituted by ²¹²Bi(²¹²Pb),²¹³Bi(III), ⁶⁴Cu(II), ⁶⁷Cu(II), ⁶⁸Ga(III), ⁸⁹Zr(IV), ^(99m)Tc(III),¹¹¹In(III), ¹⁸⁶Re(VI), ¹⁸⁸Re(VI), ²¹¹At(III), ²²⁵Ac(III), ¹⁵³Sm(III),¹⁴⁹Tb(III), ¹⁶⁶Ho(III), ²¹²Bi(²¹²Pb), ²¹³Bi(III), preferably ¹⁷⁷Lu(III),⁹⁰Y(III) and ¹⁶⁶Ho(III). Preferably, M is a radioelement chosen from theyttrium and lanthanide radioactive isotopes.

In particular, among the radioelements according to the invention,mention may be made of: ¹⁷⁷Lu, ¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ⁸⁶Y and¹⁵³Sm. According to a particular embodiment, M is a radioelement chosenfrom the group constituted by ¹⁶⁶Ho, ¹⁷⁷Lu and ⁹⁰Y.

According to one embodiment, said complex is of general formula (III)below:

in which R₁, R₂, R₃, R₄, R₅, R₆, X₁, X₂, X₃, Y₁, Y₂, Y₃ and M are asdefined above. In particular, in the general formula (III), each of thegroups Y₁, Y₂ and Y₃ comprises a C(O)O⁻ group, which allows complexingwith the element M.Pharmaceutical Composition

The invention also relates to a pharmaceutical composition comprising acompound of formula (I) as defined above or complex as defined above,and optionally one or more pharmaceutically acceptable excipients.

The composition may also comprise a buffer chosen from the buffers ofestablished use, for instance lactate, tartrate malate, maleate,succinate, ascorbate, carbonate, tris((hydroxymethyl)aminomethane),HEPES (2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid) or MES(2-morpholinoethanesulfonic acid) buffers, and mixtures thereof.

The pharmaceutical composition may comprise an oily phase, especially aniodinated oil. According to a particular embodiment, the pharmaceuticalcomposition also comprises ethyl esters of iodinated fatty acids ofpoppy oil.

According to one embodiment, the pharmaceutical composition according tothe invention is constituted by an iodinated oil and complexes accordingto the invention. Typically, the pharmaceutical composition according tothe invention is constituted by Lipiodol® and complexes according to theinvention. Lipiodol® is constituted by ethyl esters of iodinated fattyacids of poppy oil.

Preferably, the pharmaceutical composition according to the invention isradioopaque, and thus visible by x-ray radiography.

According to a particular embodiment, the pharmaceutical composition isan injectable composition. According to one embodiment, thepharmaceutical composition according to the invention is administered byintra-arterial hepatic injection.

The invention relates to a complex or a pharmaceutical composition asdefined above, for its use in the treatment of cancer.

The invention also relates to a complex or a pharmaceutical compositionas defined above, for its use in medical imaging.

The invention relates to the use of a complex as defined above for thepreparation of a medicament for treating cancer.

The invention also relates to the use of a complex or a pharmaceuticalcomposition as defined above in medical imaging.

The invention relates to a method for the therapeutic treatment of apatient suffering from cancer, comprising the administration to saidpatient of a complex or a pharmaceutical composition as defined above.In particular, said treatment method does not comprise a step ofsurgical treatment.

The invention also relates to a method for the medical imaging of atumor, comprising:

-   -   a step of administering to a patient suffering from cancer a        complex or a pharmaceutical composition according to the        invention; and    -   a step of detecting the tumor via a medical imaging method.

The term “cancer” refers to an abnormal cell proliferation (also knownas a tumor) in a normal tissue of the body. These cancer cells allderive from the same clone, a cell initiating the cancer, which hasacquired certain characteristics enabling it to divide indefinitely. Inthe course of evolution of the tumor, certain cancer cells may migratefrom their site of production and form metastases.

Among cancers, mention may be made especially of liver cancer, inparticular primary liver cancer, preferably hepatocarcinoma. Accordingto a particular embodiment, among cancers, mention may be made ofhepatocarcinoma, epitheloid hemangioendothelioma, cholangiocarcinoma,neuroendocrine tumors and metastases of other cancers such as colorectalcancer metastases.

According to a particular embodiment, the cancer is anintermediate-stage hepatocellular carcinoma, in adults.

Process for Preparing the Compounds of General Formula (I) andRadiolabeling

The compounds of general formula (I) may be prepared according to twoprocesses, depending on whether the compounds of general formula (I) aresymmetrical or dissymmetrical.

In these preparation processes, the deprotection steps are known tothose skilled in the art and correspond to standard reactions ofhydrolysis of an amide. The functionalization steps are also known tothose skilled in the art and correspond to standard alkylation reactions(cf. Loic Bellouard J CHEM S Perkin 1, (23), 1999, pages 3499-3505).

The first applicable approach for symmetrical compounds involves thetotal synthesis of the pyclene macrocycle with the need to differentiatethe central nitrogen atom (6) from the nitrogen atoms in the sidepositions (3 and 9).

To do this, the method proposed by Siauge et al. is applicable(Tetrahedron, volume 57, issue 22, pages 4713-4718). This method isbased, firstly, on the use of a nosyl (2-nitrophenylsulfonyl) groupinstead of the tosyl groups usually chosen to perform macrocyclizationsaccording to the general method of Richman and Atkins (J. Am. Chem. Soc.1974, 96, 2268-2270) and on the selective reactivity of primary aminesof diethylenetriamine. According to this principle, it is possible toprepare diethylenetriamine-based intermediates that are variouslysubstituted on the central secondary amine (prefiguring the substitutionin position 6). These compounds are key intermediates which can then beemployed in the macrocyclization reaction. The presence of nosyl groups,which are easier to deprotect than the tosyl analogs, allows theintroduction onto the macrocycle of a wider diversity of substituents inposition 6. Scheme 1 below illustrates this preparation process.

In scheme 1, X₁, X₂ and X₃ are as defined for the compounds of generalformula (I), the group Ra is a group —C(R₃)(R₄)—Y₂, with R₃, R₄ and Y₂as defined for the compounds of general formula (I), an ester or anorthogonal protecting group such as tert-butoxycarbonyl and Z is aleaving group such as Cl, Br, I, tosylate mesylate or triflate. Thispreparation process is referred to as the “Direct route” or “Boc route”in the examples.

The invention also relates to a process for preparing the compounds ofgeneral formula (I) according to the invention which are dissymmetrical.This preparation process is advantageously based on the reaction of anoxalic acid diester with pyclene, which makes it possible to block twonitrogen atoms of pyclene (N-6 and N-9) so as to be able to actselectively on the third atom (N-3) which has remained free. Afterfunctionalization of the nitrogen in position -3, deprotection of theoxalamide group leads to a pyclene which is substituted in position -3in a controlled manner, according to scheme 2 below:

In scheme 2, X₁, X₂, X₃, R₁ to R₆ and Y₁ to Y₃ are as defined for thecompounds of general formula (I). According to one embodiment, theprotection step is performed in the presence of methanol.

The invention relates to a process for preparing the compounds ofgeneral formula (I) for which the groups —C(R₁)(R₂)—Y₁ and —C(R₅)(R₆)—Y₃are different, comprising a step of functionalizing a compound ofgeneral formula (IX) below:

to form a compound of general formula (X) below:

in which X₁, X₂, X₃, R₁, R₂ and Y₁ are as defined for the compounds ofgeneral formula (I).

According to a particular embodiment, said preparation process alsocomprises:

a step of deprotection of the compound of general formula (X) to obtaina compound of general formula (XI) below:

in which X₁, X₂, X₃, R₁, R₂ and Y₁ are as defined for the compounds ofgeneral formula (I), and

-   -   a step of functionalization of the compound of general        formula (XI) to obtain a compound of general formula (I) as        defined above.

The term “functionalization” means the addition to a nitrogen atom of agroup —C(R₁)(R₂)—Y₁, —C(R₃)(R₄)—Y₂ or —C(R₅)(R₆)—Y₃.

The invention also relates to a compound of general formula (X) below:

in which X₁, X₂, X₃, R₁, R₂ and Y₁ are as defined for the compounds ofgeneral formula (I).

According to a particular embodiment of the process for preparing thedissymmetrical compounds of formula (I), when at least one of the Ri isother than H, the preparation of a substituted picolinate intermediateis performed via a bromo derivative in position -4, which makes itpossible, via a palladium-catalyzed coupling reaction with an alkyne(Sonogashira reaction, Comprehensive Chirality, volume 4, pages 18-32,2012), to install the chosen residue, according to scheme 3 below(example with a C₁₂ alkyne):

The invention also relates to a process for radiolabeling the compoundsof general formula (I), said radiolabeling process preferably beingperformed at a pH of between 6.5 and 9. According to a particularembodiment, said radiolabeling is performed in the presence of acetatebuffer. According to one embodiment, the radiolabeling is performed inthe presence of water or of an alcohol such as ethanol, or mixturesthereof.

According to another embodiment, the radiolabeling is performed at atemperature of between 80° C. and 100° C.

DESCRIPTION OF THE FIGURES

FIG. 1: ¹H NMR spectra of the ligand P04213 and of its yttrium complexP04183 (300 MHz, 298 K, D₂O)

FIG. 2: Absorption spectra of the ligands and of their yttrium complexesrecorded in water at pH 3.8 and 5.5 (acetate buffer). The absorptionband corresponding to the π-π* transitions of pyridine extends from 240to 300 nm for the ligands and the complexes.

FIG. 3: Monitoring of the variation in absorbance at the Amax of thecomplex or of the ligand at pH 5.5 and 3.8.

FIG. 4: ¹H NMR spectrum of the ligand P04330.

FIG. 5: Percentage of extraction of the complex P04283 with ⁹⁰Y as afunction of time, in hours (Re-SSS is a reference complex).

FIG. 6: Ratio between the relaxivity of the gadolinium complexes ofligands P04218 and P04216 measured at a given time and that at t=0 minas a function of time in the presence of a solution of Zn andphosphates.

The examples that follow are described as illustrations of the presentinvention.

EXAMPLES

Summary table of the various names of the specific compounds of generalformula (I) according to the invention:

Yttrium ⁹⁰Yttrium Abbreviation Ligand complex complex Mono S Pc-2a1paSym P04218 P04219 Mono AS Pc-2a1pa Asym P04216 P04217 Di Sym Pc-1a2pasym P04213 P04183 Di AS Pc-1a2pa Asym P04214 P04215 P04233 Tri Pc-3paP04221 P04222 Di AS Pc-1a2pa Asym P04245 P04283 C12 Di AS Pc-1a2pa AsymP04330 C8

A—Materials and Methods

Yttrium-90 chloride is purchased from PerkinElmer Life Sciences. Theactivities involved were between 28 μCi and 8.51 mCi (1.04-314.87 MBq).The products (HPLC solvents, buffers, etc.) are used as furnished,without further purification. Unless otherwise specified, the ligand isdissolved in ethanol.

The experiments were performed in crimped borosilicate glass bottles.The bottles were heated in a Bioblock heating block allowing up to 6bottles to be heated. When stirring was necessary, a Lab Dancer S40(VWR) vortex machine was used. The centrifugations were performed withan MF 20-R centrifuge (Awel).

The activities were measured in a CRC-127R activimeter (Capintec), whichwas calibrated each morning.

The quality controls were performed by TLC on Whatman 1 paper with anMeOH/0.1% Et₃N mixture as eluent. The radiochemical purities aredetermined using a Cyclone phosphoimager (Perkin Elmer), with the aid ofthe Optiquant software.

HPLC analyses were also performed, on a Dionex Ultimate 3000 HPLC lineequipped with a diode array detector and an fLumo radiochromatographicdetector (Berthold), run by the Chromeleon software.

The analyses were performed on an Accucore C18 100×3 mm, 2.6μ columnwith the following program: 0.4 mL/min; A=H₂O; B=ACN; 0-3 min: 100% A;3-20 min: 0-90% B; 20-25 min: 10% A/90% B; 25-26 min: 90-0% B; 26-30min: 100% A.

Spectroscopic Studies

The UV-visible spectra of the ligands and of the yttrium(III) complexeswere measured in aqueous solution of acetate buffer (pH=5.5 or 3.8without control of the ionic strength) at 298 K using a Jasco V-650spectrophotometer.

The NMR experiments (COSY, HMBC and HMQC) were recorded for the ligandsand their complexes using a Brüker Avance 500 spectrometer (500 MHz) inD₂O.

Kinetic Studies

The formation of the yttrium(III) complexes of do2pa sym, do2pa asym anddo1pa sym were studied in an aqueous solution of acetate buffer (C=0.150M) at 25° C. under pseudo-first-order conditions. The increase inintensity of the absorption band in the UV region was monitored atpH=3.8 and 5.5 with C_(L)=C_(M)=4×10⁻⁵ M and without control of theionic strength.Dissociation in acidic medium of the yttrium(III) complexes was studiedunder pseudo-first-order conditions without control of the ionicstrength and by addition of aqueous solutions of HCl (0.5, 1, 2, 4 and5M) to a solution of complex (C=4.10⁻⁵ M).The dissociation was monitored by decrease of the intensity of theabsorption band of the complex or increase of the absorption band of theligand in the UV region. t_(1/2) was calculated by adjusting the curveA_(max)=f (t) (A_(max)=absorbance at λ_(max) of the complex or of theligand) with the following pseudo-first-order exponential equation:Abs(t)=Abs(eq)+(Abs(0)−Abs(eq))×exp (−x/t1).Potentiometric StudiesEquipment:

The experiments were performed under an inert atmosphere in aqueoussolutions thermostatically maintained at 25.0±0.1° C. The protonationand complexation titrations were performed in a jacketed glasstitrations cell using a Metrohm 702 SM Titrino automatic buretteconnected to a Metrohm 6.0233.100 combined glass electrode. Thetitrations were controlled automatically by software after selection ofthe appropriate parameters avoiding monitoring during long measurements.

The titrant is an approximately 0.1 M KOH solution prepared from ananalytical-grade commercial vial and its exact concentration is obtainedby applying the Gran method by titrating with a standard HNO₃ solution.

The ligand solutions were prepared at approximately 2.0×10⁻³ M and theCu²⁺, Pb²⁺ and Y³⁺ solutions at approximately 0.04 M fromanalytical-grade chloride salts and standardized by complexometrictitration with H₄edta (ethylenediaminetetraacetic acid)¹. The solutionto be titrated contains approximately 0.05 mmol of ligand in a volume of30.00 mL, the ionic strength of which was maintained at 0.10 M usingKNO₃ as electrolyte. 1.2 equivalents of metal cation (Cu²⁺ or Pb²⁺) wereadded to the ligand (0.05 mmol) during the standardization titrations ofthe ligand solution.0.9 equivalent of metal cation (Y³⁺) was added to the ligand during thecomplexation titration methods.Measurements

The electromotive force of the solution was measured after calibrationof the electrode by titration of a standard 2.10⁻³ M HNO₃ solution. The[H⁺] of the solutions was determined by measuring the electromotiveforce of the cell, E=E°′+Q log [H⁺]+Ej. The term “pH” is defined by−log[H⁺]. E°′ and Q are determined by the acidic region of thecalibration curves. The liquid junction potential, Ej, is negligibleunder the experimental conditions used. The value of K_(e)=[H⁺][OH⁻] is10^(−13.78).

Calculations

The potentiometric data were refined with the Hyperquad software² andthe speciation diagrams were plotted using the HySS software³.

The overall equilibrium constants β_(i)H and βM_(m)H_(h)L_(l) aredefined by βM_(m)H_(h)L_(l)=[M_(m)H_(h)L_(l)]/[M]_(m)[H]_(h)[L]_(l)(β_(i)H=[H_(h)L_(l)]/[H]_(h)[L]_(l) and βMH⁻¹L=βML(OH)×Ke). Thedifferences, in log units, between the protonation (or hydrolysis)values and the non-protonation constants give the intermediate reactionconstants (log K) (withKM_(m)H_(h)L_(l)=[M_(m)H_(h)L_(l)]/[M_(m)H_(h−1)L_(l)][H]). The errorsindicated are the standard deviations calculated by the adjustmentprogram from all of the experimental data for each system.

B— Synthesis of the Compounds of General Formula (I)

All the commercial reagents were used as received from the suppliers,unless otherwise indicated. The solvents were distilled before use,according to the procedures described in the literature. Thepurifications by semi-prep HPLC (high-performance liquid chromatography)were performed with a Prominence Shimadzu HPLC/LCMS-2020 machineequipped with an SPD-20 A UV detector. The HPLC chromatographic systemuses a column (VisionHT C₁₈ HL 5μ250×10 mm) eluted with an H₂O (with0.1% TFA or HCl)-MeCN isocratic gradient.The ¹H and ¹³C NMR spectra were recorded on a Brüker AMX3-300 MHzspectrometer operating at 300.17 and 75.47 MHz, respectively, for ¹H and¹³C. All the measurements were taken at 25° C. The signals are indicatedas follows: δ chemical shift (ppm), multiplicity (s, singlet; d,doublet; t, triplet; m, multiplet; q, quartet), integration, couplingconstants J in hertz (Hz).The high-resolution mass spectrometry (HRMS-ESI) was performed inpositive electrospray ionization mode (ESI+) by the mass spectrometrydepartment of the Institut de Chimie Organique et Analytique (ICOA),Orleans, France.

1) Synthesis of the Ligands Pc1a2pa Sym P04213 of Formula (I) Via the“Direct” Route

Example 1—Int.2

A solution of 2-nitrobenzenesulfonyl chloride (4.3 g, 19.4 mmol) infreshly distilled THF is added at 0° C. to a mixture ofdiethylenetriamine (1.0 g, 9.69 mmol) and NaHCO₃ (3.26 g, 38.8 mmol) inTHF (200 ml). The medium is stirred at room temperature for 20 hours andthe solid is then filtered off. The filtrate is concentrated to drynessto give a white solid. This compound is used in the following reactionwithout purification.

¹H NMR (300 MHz, DMSO-d₆): δ 8.03-7.82 (m, 8H), 2.87 (t, 4H, ³J=6.0 Hz),2.47 (t, 4H, ³J=6.0 Hz).

¹³C NMR (75.47 MHz, DMSO-d₆): δ 147.73, 133.99, 132.68, 132.60, 129.49,124.39, 47.80, 42.65.

Example 1—Int.3

A solution of tert-butyl bromoacetate (6.09 g, 31.2 mmol) in THF (50 ml)is added to a solution of the compound prepared previously (4.93 g, 10.4mmol) and of triethylamine (6.31 g, 62.4 mmol) in THF (75 ml). Themixture is refluxed for 24 hours. After cooling the medium, 50 ml ofsaturated NH₄Cl solution are added and the solvent is removed byevaporation under reduced pressure. The aqueous phase thus obtained isextracted three times with 50 ml of CH₂Cl₂. The chloromethylenefractions are combined and dried over MgSO₄ and then filtered. Afterevaporating off the solvent, the white solid obtained is chromatographedon silica gel (5/5 to 8/2 ethyl acetate/pentane) to give a white solidafter evaporating off the solvent (2.9 g, 51% calc. starting from 1).

¹H NMR (300 MHz, CDCl₃): δ 8.09 (m, 2H), 7.82 (m, 2H), 7.72 (m, 4H),5.94 (t, 2H, ³J=5.7 Hz), 3.17 (s, 2H), 3.07 (m, 4H), 2.76 (t, 4H, ³J=5.7Hz), 1.41 (s, 9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 170.78, 148.27, 133.71, 133.45, 132.78,130.97, 125.52, 82.13, 55.99, 54.65, 41.90, 28.16.

Example 1—Int.4

4 g of K₂CO₃ are added to a solution in acetonitrile (20 ml) of thecompound prepared previously (2.86 g, 4.87 mmol) and the mixture isbrought to reflux. Dibromomethylpyridine (1.55 g, 5.84 mmol) dissolvedin 10 ml of acetonitrile is then added. The medium is refluxed overnightand the solid is filtered off after cooling. The solvent is evaporatedoff under reduced pressure. The compound obtained is used in thefollowing reaction without further purification.

¹H NMR (300 MHz, CDCl₃): δ 8.1-7.6 (m, 9H), 7.42 (d, 2H, ³J=7.8 Hz),4.56 (s, 4H), 3.30 (m, 4H), 3.17 (s, 2H), 2.57 (m, 4H), 1.37 (s, 9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 171.09, 154.75, 148.33, 139.17, 133.81,132.74, 131.91, 130.86, 124.39, 124.27, 81.25, 57.51, 54.33, 51.18,44.93, 28.18.

Example 1—Int.5

The compound prepared previously (2.9 g, 4.29 mmol) is dissolved in DMFin the presence of Na₂CO₃ (3.64 g, 34.3 mmol). Thiophenol (1.88 g, 17.2mmol) is then added and the medium is stirred at room temperatureovernight. After evaporating off the solvent, the residue is taken up inCH₂Cl₂ (100 ml) and the solution obtained is washed with 3×40 ml of 0.5M NaOH solution. After drying over MgSO₄ and filtration, the organicsolution is concentrated and the product obtained is chromatographed onneutral alumina (99/1 CH₂Cl₂/MeOH) to give a white solid (0.835 g, 54%calc. starting from 3).

¹H NMR (300 MHz, CDCl₃): δ 7.4 (t, 1H, ³J=7.5 Hz), 6.86 (d, 2H, ³J=7.5Hz), 3.83 (s, 4H), 3.22 (s, 2H), 2.48 (m, 4H), 2.40 (m, 4H), 1.28 (s,9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 171.06, 157.49, 136.65, 120.03, 80.98,59.10, 56.00, 52.67, 47.41, 27.95.

Example 1—Int.6

The methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid (0.872 g,4.70 mmol) is added to a solution of the compound prepared previously(0.835 g, 2.61 mmol) in acetonitrile (35 ml) in the presence of K₂CO₃(1.4 g, 10.4 mmol). The medium is refluxed overnight and then filteredand concentrated. The residue is purified by chromatography on neutralalumina (98/2 CH₂Cl₂/MeOH) to give 1.09 g of a yellow oil (67%).

¹³C NMR (75.47 MHz, CDCl₃): δ 169.95, 164.98, 158.48, 145.96, 138.30,137.54, 126.69, 123.11, 119.93, 81.07, 62.45, 61.81, 54.44, 53.13,52.18, 51.19, 27.51.

Example 1—Int.7

The compound prepared previously (1.09 g, 1.76 mmol) is dissolved in 6Mhydrochloric acid solution and the medium is refluxed overnight. Afterconcentration, the product is purified by HPLC on a C₁₈ phase (100/0 to10/90 H₂O/acetonitrile) to give intermediate 7 in hydrochloride form(0.690 g, 57% calc. for 3 HCl).

¹H NMR (500.25 MHz, D₂O): δ 8.21 (t, 2H, ³J=7.8 Hz), 8.07 (d, 2H, ³J=7.8Hz), 7.88 (d, 2H, ³J=7.8 Hz), 7.68 (t, 1H, ³J=7.8 Hz), 7.07 (d, 2H,³J=7.8 Hz), 4.63 (s, 4H), 4.45 (s, br, 4H), 3.78 (s, 2H), 3.58 (m, 4H),3.46 (s, br, 4H).

¹³C NMR (125.79 MHz, D₂O): δ 175.11, 170.44, 157.33, 153.78, 152.13,145.76, 142.12, 131.02, 127.79, 124.63, 62.55, 60.72, 57.63, 56.20,54.67.

2) Synthesis of Pc2a1pa Sym P04218 of Formula (I) Via the “Direct” Route

Example 2—Int.8

The methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid (1.93 g,10.42 mmol) is added to a solution of compound 2 (Example 1-int.2)(4.935 g, 10.42 mmol) in acetonitrile (60 ml) in the presence of K₂CO₃(4.3 g, 31.26 mmol) and the mixture is stirred at room temperature for 4days. The solvent is evaporated off and the residue is taken up inCH₂Cl₂, filtered, concentrated and purified by chromatography on silicagel (5/5 to 8/2 ethyl acetate/pentane). The product is recovered in theform of a yellow oil (2.78 g, 46% calc. starting from 1).

¹H NMR (300 MHz, CDCl₃): δ 8.08-7.99 (m, 2H), 7.95 (d, 1H, ³J=7.9 Hz),7.82-7.61 (m, 7H), 7.47 (d, 1H, ³J=7.9 Hz), 6.21 (m, 2H), 3.94 (s, 3H),3.78 (s, 2H), 3.10 (m, 4H), 2.68 (t, 4H, ³J=5.5 Hz).

¹³C NMR (75.47 MHz, CDCl₃): δ 165.64, 159.19, 148.11, 147.80, 138.01,133.58, 132.71, 130.71, 126.16, 125.18, 123.99, 59.45, 54.83, 53.03,41.58.

Example 2—Int.9

The compound prepared previously (2.78 g, 4.46 mmol) and 3.7 g of K₂CO₃in 30 ml of acetonitrile are brought to reflux and a solution ofdibromomethylpyridine (1.42 g, 5.36 mmol) in 10 ml of acetonitrile isthen added. The medium is stirred at the reflux point of theacetonitrile overnight, the solid is then filtered off and the filtrateis concentrated under reduced pressure. The compound obtained is used inthe following step without further purification.

¹H NMR (300 MHz, CDCl₃): δ 7.88-7.50 (m, 12H), 7.35 (d, 2H, ³J=7.5 Hz),4.52 (s, 4H), 3.89 (s, 3H), 3.77 (s, 2H), 3.27 (m, 4H), 2.52 (m, 4H).

¹³C NMR (75.47 MHz, CDCl₃): δ 165.23, 159.52, 153.98, 147.55, 146.63,138.66, 137.10, 133.39, 131.81, 131.45, 130.08, 125.18, 123.78, 123.57,123.25, 120.99, 120.56, 59.82, 53.62, 52.33, 48.61, 43.35.

Example 2—Int.10

The compound prepared previously (3.9 g, 5.5 mmol) is dissolved in DMFin the presence of Na₂CO₃ (4.6 g, 43.9 mmol), thiophenol (2.42 g, 21.9mmol) is then added and the medium is stirred at room temperatureovernight. The solvent is then removed by distillation under reducedpressure and the residue is taken up in 100 ml of CH₂Cl₂. After washingthe organic phase 3 times (3×40 ml) with 0.5M sodium hydroxide solution,drying over MgSO₄ and evaporation of the solvent, the residue obtainedis purified by chromatography on neutral alumina (99/1 CH₂Cl₂/MeOH) togive a yellow oil (0.297 g, 19% calc. starting from 8).

¹H NMR (300 MHz, CDCl₃): δ 7.93 (d, 1H, ³J=7.5 Hz), 7.76 (t, 1H, ³J=7.5Hz), 7.61 (t, 1H, ³J=7.5 Hz), 7.40 (d, 1H, ³J=7.5 Hz), 7.07 (d, 2H,³J=7.5 Hz), 4.13 (s, 4H), 4.00 (s, 2H), 3.79 (s, 3H), 2.73 (m, 8H).

¹³C NMR (75.47 MHz, CDCl₃): δ 165.32, 159.60, 155.26, 147.50, 137.96,137.52, 125.85, 123.96, 120.60, 61.47, 55.96, 52.67, 51.96, 47.09.

Example 2—Int.11

To a mixture of tert-butyl bromoacetate (0.294 g, 1.50 mmol) and K₂CO₃(0.464 g, 3.4 mmol) in 10 ml of acetonitrile is added the compoundprepared previously (0.297 g, 0.84 mmol). The medium is refluxedovernight, the solid is then filtered off and the solution obtained isconcentrated. The residue obtained is purified by chromatography onneutral alumina (100/0 to 95/5 CH₂Cl₂/MeOH) to give compound 11 in theform of a yellow oil (0.185 g, 38%).

Example 2—Int.12

The compound prepared previously (0.185 g, 0.32 mmol) is dissolved in 20ml of 6M HCl and the medium is refluxed overnight. After evaporating offthe solvent, the product is purified by HPLC on a C18 phase (100/0 to10/90 H₂O/acetonitrile) to give the expected product in hydrochlorideform (0.050 g, 27% calc. for 3 HCl).

¹H NMR (500.25 MHz, D₂O): δ 8.33 (t, 1H, ³J=7.8 Hz), 8.25 (d, 1H, ³J=7.8Hz), 8.07 (d, 1H, ³J=7.8 Hz), 8.00 (t, 1H, ³J=7.8 Hz), 7.49 (d, 2H,³J=7.8 Hz), 4.81 (s, 4H), 4.20 (s, 2H), 3.76 (s, 4H), 3.63 (m, 4H), 2.99(s, br, 4H).

¹³C NMR (125.79 MHz, D₂O): δ 172.17, 168.64, 157.78, 152.89, 150.25,146.36, 142.80, 131.30,

3) Synthesis of a Preparation Intermediate Compound

Example 3—Int.3′

To a solution of compound 2 (Example 1-int.2) and of triethylamine (3.9g, 38.8 mmol) in freshly distilled THF (150 ml) is added, at 0° C., asolution of di-tert-butyl dicarbonate (5.07 g, 23.3 mmol) in freshlydistilled THF (50 ml). The medium is stirred at room temperature for 24hours and then treated with saturated NH₄Cl solution. The solvent isevaporated off under reduced pressure and the aqueous phase is washedwith dichloromethane (3×80 ml). After drying over MgSO₄, the organicsolution is filtered and concentrated under vacuum. The residue ischromatographed on silica gel (3/7 to 7/3 ethyl acetate/heptane) to givethe expected compound in the form of a yellow oil (7.0 g, 79%).

¹H NMR (300 MHz, CDCl₃): δ 8.05-7.63 (m, 2H), 7.79-7.63 (m, 6H), 5.99(s, br, 1H), 5.77 (s, br, 1H), 3.3 (m, 4H), 3.19 (m, 4H), 1.37 (s, 9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 155.78, 147.75, 133.77, 133.09, 132.95,130.71, 125.20, 80.85, 42.34, 28.16.

Example 3—Int.4′

To a mixture composed of the product prepared previously (7.0 g, 12.2mmol), Na₂CO₃ and DMF (200 ml) heated to 100° C. is added, under anitrogen atmosphere, a solution of 2,6-bis(bromomethyl)pyridine (3.23 g,12.2 mmol) in 100 ml of dry DMF. The medium is stirred at 100° C. for 24hours and then cooled. The solvent is evaporated off under reducedpressure and the residue thus obtained is taken up in CH₂Cl₂. Theorganic phase is washed with 1M NaOH solution and dried with MgSO₄.After filtration and concentration, the product is precipitated fromacetone to give a white solid (3.76 g, 46%).

¹H NMR (300 MHz, DMSO-d₆): δ 8.10-7.80 (m, 9H), 7.35 (m, 2H), 4.60 (s,4H), 3.53 (s, 8H), 1.38 (s, 9H).

¹³C NMR (75.47 MHz, DMSO-d₆): δ 155.83, 155.75, 154.59, 147.95, 147.89,138.40, 135.63, 134.57, 132.75, 132.62, 131.17, 130.97, 129.52, 129.19,124.62, 124.63, 122.49, 122.44, 78.81, 55.17, 50.02, 49.79, 45.42,44.72, 44.66, 43.09, 27.97.

Example 3-Int.5′

To a suspension of Na₂CO₃ in 250 ml of DMF are added the compoundprepared previously (3.59 g, 5.43 mmol) and then thiophenol (2.35 g,21.3 mmol). The mixture is stirred at room temperature for 12 hours, thesolvent is then evaporated off under reduced pressure and the residueobtained is taken up in CH₂Cl₂. The organic phase is washed with water,dried over MgSO₄ and then filtered and concentrated. The residue thusobtained is purified by chromatography on silica gel (100/0 to 95/5MeOH/32% NH₃aq) to give the expected compound in the form of a yellowoil (1.06 g, 76%).

¹H NMR (300 MHz, CDCl₃): δ 7.51 (t, 1H, ³J=7.5 Hz), 6.94 (d, 2H, ³J=7.5Hz), 3.94 (s, 4H), 3.52 (t, 4H, ³J=5.1 Hz), 3.04 (s, 2H), 2.61 (t, 4H,³J=5.65 Hz), 1.49 (s, 9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 158.22, 157.53, 136.65, 120.34, 80.27,52.06, 50.78, 48.88, 28.59.

4) Synthesis of the Ligand Pc2a1pa Sym P04218 of Formula (I) Via the“Boc” Route

Example 4-Int.6′

To a mixture composed of the product obtained previously (Example3-int.5′) (0.803 g, 2.62 mmol) and K₂CO₃ in 150 ml of acetonitrile isadded a solution of tert-butyl bromoacetate (1.022 g, 5.24 mmol) in 50ml of acetonitrile and the mixture is stirred at room temperature for 24hours. The solvent is evaporated off, the residue is taken up in CH₂Cl₂and the solution obtained is then filtered and concentrated. The productis purified by chromatography on silica gel (100/0 to 98/2 CH₂Cl₂/MeOH)to give a yellow oil (1.06 g, 76%).

¹H NMR (300 MHz, CDCl₃): δ 7.5 (t, 1H, ³J=7.5 Hz), 7.08 (d, 2H, ³J=7.5Hz), 3.86 (s, br, 4H), 3.27 (d, 4H, ³J=9.4 Hz), 3.01 (m, 4H), 2.75-2.55(m, 4H), 1.34 (s, 18H), 1.24 (s, 9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 170.45, 170.27, 157.44, 156.97, 155.26,137.18, 122.67, 122.61, 80.75, 78.71, 59.99, 59.60, 59.02, 58.67, 51.77,51.27, 45.04, 44.81, 28.21, 28.03.

Example 4-Int.7′

The compound prepared previously (1.06 g, 9.5 mmol) is dissolved in 20ml of 6M hydrochloric acid and the mixture is refluxed overnight. Aftercooling, the solvent is evaporated off and the expected product isobtained in the form of a brown solid (100%).

¹H NMR (300 MHz, D₂O): δ 7.91 (t, 1H, ³J=7.9 Hz), 7.36 (d, 2H, ³J=7.9Hz), 4.20 (s, 4H), 3.65 (s, 4H), 2.96 (m, 4H), 2.78 (m, 4H).

¹³C NMR (75.47 MHz, D₂O): δ 175.61, 154.59, 147.87, 127.29, 60.26,59.52, 54.14, 46.69.

Example 4-Int.8′

To a solution of the compound obtained previously in 30 ml of methanolis added 5 ml of concentrated H₂SO₄ and the mixture is then stirred andrefluxed overnight. After cooling, the solvent is evaporated off, theresidue is taken up in 10 ml of water and the pH is adjusted to 7 byadding K₂CO₃. The water is evaporated off and the residue is taken up indichloromethane. The organic phase is then dried over MgSO₄, filteredand concentrated. The expected product is obtained in the form of yellowoil (0.67 g, 98% calc. starting from 6′).

¹H NMR (300 MHz, CDCl₃): δ 7.34 (t, 1H, ³J=7.5 Hz), 6.84 (d, 2H, ³J=7.5Hz), 3.86 (s, 4H), 3.51 (s, 10H), 2.69 (m, 4H), 2.02 (m, 4H).

¹³C NMR (75.47 MHz, CDCl₃): δ 172.12, 159.24, 136.56, 120.66, 59.54,57.73, 52.59, 50.95, 46.99.

Example 4-Int.9′

To a mixture composed of the product obtained previously (0.67 mg, 1.9mmol) and K₂CO₃ (0.524 g, 3.8 mmol) in 50 ml of acetonitrile is added0.353 g (1.9 mmol) of the methyl ester of6-chloromethyl-2-pyridinecarboxylic acid and the medium is stirred fortwo days at room temperature. The solvent is evaporated off and theresidue is taken up in CH₂Cl₂ and then filtered. The solution obtainedis concentrated and the product is used directly in the following stepwithout further purification.

Example 4-Int.12

To the compound prepared previously are added 20 ml of 6M hydrochloricacid and the mixture is refluxed overnight. The solvent is evaporatedoff and the residue obtained is purified by HPLC on a C18 phase (100/0to 10/90 H₂O 0.1% HCl/acetonitrile) to give the expected product in theform of a colorless oil (0.237 g, 22% calc. starting from 8′ for 3 HCl).

¹H NMR (500.25 MHz, D₂O): δ 8.33 (t, 1H, ³J=7.8 Hz), 8.25 (d, 1H, ³J=7.8Hz), 8.07 (d, 1H, ³J=7.8 Hz), 8.00 (t, 1H, ³J=7.8 Hz), 7.49 (d, 2H,³J=7.8 Hz), 4.81 (s, 4H), 4.20 (s, 2H), 3.76 (s, 4H), 3.63 (m, 4H), 2.99(s, br, 4H).

¹³C NMR (125.79 MHz, D₂O): δ 172.17, 168.64, 157.78, 152.89, 150.25,146.36, 142.80, 131.30, 128.33, 125.60, 62.35, 60.08, 59.47, 56.09,52.88.

5) Synthesis of the Ligand Pc1a2pa Sym P04213 of Formula (I) Via the“Boc” Route

Example 5-Int.11′

To a mixture composed of the product obtained previously (Example3-int.5′) (0.326 g, 1.06 mmol) and K₂CO₃ (0.587 g, 4.3 mmol) in 50 ml ofacetonitrile is added a solution of the methyl ester of6-chloromethyl-2-pyridinecarboxylic acid (0.395 g, 2.13 mmol) in 20 mlof acetonitrile. The mixture is stirred at room temperature for 5 daysand the solvent is evaporated off. The residue is taken up indichloromethane and the suspension is filtered. The chloromethylenesolution is concentrated and the residue is purified by chromatographyon neutral alumina (100/0 to 98/2 CH₂Cl₂/MeOH) to give a yellow oil(0.407 g, 63%).

¹H NMR (300 MHz, CDCl₃): δ 8.05-7.95 (m, 2H), 7.87-7.73 (m, 4H), 7.66(t, 1H, ³J=7.2 Hz), 7.2 (m, 2H), 4.10-3.80 (m, 14H), 3.46-3.31 (m, 4H),2.75-2.50 (m, 4H), 1.17 (s, 9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 165.91, 160.82, 160.70, 156.80, 156.54,155.48, 147.44, 137.66, 137.55, 137.38, 126.14, 126.07, 123.83, 23.14,122.96, 79.03, 62.90, 62.71, 59.96, 58.78, 53.00, 51.59, 51.27, 45.14,44.75, 28.30.

Example 5-Int.12′

To a solution of the compound prepared previously (0.407 g, 0.67 mmol)in 20 ml of methanol is added 1 ml of concentrated sulfuric acid. Themixture is stirred at reflux for 2 days. After cooling, the solvent isevaporated off, the residue is taken up in water (10 ml) and the pH ofthe medium is adjusted to 7 by adding K₂CO₃. The water is evaporated offand the residue is taken up in dichloromethane. The organic phase isdried over magnesium sulfate, filtered and concentrated. The product ispurified by chromatography on neutral alumina (100/0 to 98/2CH₂Cl₂/MeOH) to give a yellow oil (0.214 g, 63%).

¹³C NMR (75.47 MHz, CDCl₃): δ 165.60, 159.30, 159.24, 146.64, 137.17,127.10, 123.58, 119.79, 61.92, 57.51, 52.72, 52.56, 46.12.

Example 5-Int.6

To a mixture of the compound prepared previously (0.214 g, 0.423 mmol)and K₂CO₃ (0.117 g, 0.85 mmol) in 20 ml of acetonitrile is added asolution of tert-butyl bromoacetate (0.083 g, 0.423 mmol) in 10 ml ofacetonitrile. The mixture is stirred at room temperature for 24 hoursand then concentrated. The residue is taken up in CH₂Cl₂ and the saltsare filtered off. After evaporating off the solvent, the residue ispurified by chromatography on neutral alumina (100/0 to 98/2CH₂Cl₂/MeOH) to give the expected product in the form of a yellow oil(0.155 g, 60%).

Example 5-Int.7

The compound obtained previously is dissolved in 20 ml of 6Mhydrochloric acid and the mixture is refluxed overnight. Afterevaporating off the water, the residue is purified by HPLC on a C₁₈phase (100/0 to 90/10 H₂O/ACN) to give a colorless oil (0.089 g, 55%calc. for 3 HCl).

¹H NMR (500.25 MHz, D₂O): δ 8.21 (t, 2H, ³J=7.8 Hz), 8.07 (d, 2H, ³J=7.8Hz), 7.88 (d, 2H, ³J=7.8 Hz), 7.68 (t, 1H, ³J=7.8 Hz), 7.07 (d, 2H,³J=7.8 Hz), 4.63 (s, 4H), 4.45 (s, br, 4H), 3.78 (s, 2H), 3.58 (m, 4H),3.46 (s, br, 4H).

¹³C NMR (125.79 MHz, D₂O): δ 175.11, 170.44, 157.33, 153.78, 152.13,145.76, 142.12, 131.02, 127.79, 124.63, 62.55, 60.72, 57.63, 56.20,54.67.

REFERENCES

-   1. Schwarzenbach, G.; Flaschka, W. Complexometric Titrations;    Methuen & Co.: London, 1969.-   2. Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739-1753.-   3. Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.;    Vacca, A. Coord. Chem. Rev. 1999, 184,    -   311-318.    -   6) Synthesis of the Ligand Pc1a2pa Asym P04214 of Formula (I)        Via the “Oxalate” Route

solution of diethyl oxalate (2.02 g, 13.8 mmol) in EtOH (100 mL) wasadded to a solution of pyclene (2.37 g, 11.5 mmol) in EtOH (200 mL). Themixture was stirred at room temperature overnight and then concentrated.The residue obtained was purified by chromatography on a column ofalumina (98/2 CH₂Cl₂/MeOH). The final product was obtained in the formof a white solid (0.548 g, 19%).

¹H NMR (300 MHz, CDCl₃): δ 7.52 (t, 1H, ³J=7.7 Hz), 7.02 (d, 1H, ³J=7.9Hz), 6.93 (d, 1H, ³J=7.5 Hz), 5.59 (d, 1H, ²J=16.2 Hz), 4.62 (ddd, 1H,²J=13.9 Hz, ³J=11.1 Hz, ³J=2.5 Hz), 4.08 (d, 1H, ²J=16.6 Hz), 3.95 (d,1H, ²J=17.3 Hz), 3.77 (ddd, 1H, ²J=13.9 Hz, ³J=10.6 Hz, ³J=4.52 Hz),3.70 (d, 1H, ²J=17.3 Hz), 3.5 (ddd, 1H, ²J=12.4 Hz, ³J=10.6 Hz, ³J=4.5Hz), 3.24 (dt, 1H, ²J=13.9 Hz, ³J=4.4 Hz), 3.13 (dt, 1H, ²J=12.4 Hz,³J=4.1 Hz), 3.01 (dt, 1H, ²J=12.2 Hz, ³J=3.2 Hz), 2.83 (dt, 1H, ²J=13.9Hz, ³J=3.0 Hz), 2.74 (td, 1H, ²J=11.7 Hz, ³J=2.3 Hz).

¹³C NMR (75.47 MHz, CDCl₃): δ 162.96, 161.23, 159.10, 153.42, 136.83,120.58, 119.44, 55.40, 52.53, 47.89, 47.66, 44.61, 44.20

Synthesis of the Pyclene Oxalate Intermediate 2″:

Various tests were performed to obtain the “pyclene oxalate”intermediate 2″, as indicated below.

Route Test used Conditions Purification Yield 1 3 DIPEA, EtOH, 2Precipitation 22% days, RT 2 1 EtOH, 1.5 days, Chromatography on 33% RTalumina 3 1 EtOH, 2 days, RT Chromatography on 36% alumina 4 2 EtOH, 41h, RT Chromatography on 37% alumina 5 1 MeOH, 48 h, RT Precipitation 54%6 1 MeOH, 23 h, RT Precipitation 93% Summary of the tests on thesynthesis of the pyclene oxalate intermediate 2″

It is observed that the “pyclene oxalate” intermediate 2″ is obtainedaccording to the various operating conditions and routes tested. Inparticular, a very good yield is observed, greater than 90%, in thepresence of methanol.

Continuation of the Synthesis:

A solution of tert-butyl bromoacetate (0.668 g, 3.42 mmol) inacetonitrile (100 mL) was added to a solution of 2″ (0.890 g, 3.42 mmol)and K₂CO₃ (1.42 g, 10.3 mmol) in acetonitrile (150 mL). The mixture wasstirred at room temperature for 24 hours. The solvent was evaporated offand the residue was taken up in dichloromethane and then filtered andconcentrated. The desired product was obtained in the form of a yellowoil and used in the following steps without further purification (1.25g).

¹H NMR (300 MHz, CDCl₃): δ 7.28 (t, 1H, ³J=7.7 Hz), 6.8 (d, 1H, ³J=7.5Hz), 6.63 (d, 1H, ³J=7.5 Hz), 5.26 (d, 1H, ²J=16.6 Hz), 4.08 (m, 1H),3.89 (d, 1H, ²J=16.6 Hz), 3.68 (m, 4H), 3.0 (m, 4H), 2.77 (m, 2H), 2.52(m, 1H), 1.18 (m, 9H).

¹³C NMR (75.47 MHz, CDCl₃): δ 170.65, 162.42, 159.73, 158.43, 153.60,136.48, 119.67, 119.11, 80.37, 61.08, 56.45, 52.59, 52.07, 46.64, 46.07,44.76, 27.61.

Compound 3″ was dissolved in MeOH (100 mL) and concentrated sulfuricacid (10 mL) was added slowly. The mixture was refluxed for 24 hours.After cooling to room temperature, the solvent was evaporated off. 20 mLof water were added and the pH was adjusted to 7 with K₂CO₃. The waterwas evaporated off and the residue was taken up in dichloromethane.Magnesium sulfate was added and the organic phase was filtered and thenconcentrated. The crude product was purified by chromatography on acolumn of alumina (98/2 to 95/5 CH₂Cl₂/MeOH). Compound 4″ is in the formof a white solid (0.939 g, 99% calculated starting from 2″).

¹H NMR (300 MHz, CDCl₃): δ 7.52 (t, 1H, ³J=7.5 Hz), 6.96 (m, 2H), 3.96(d, 4H, ³J=9.8 Hz), 3.63 (m, 5H), 3.28 (m, 2H), 3.18 (m, 2H), 2.86 (dt,4H, ²J=11.3, ³J=5.7 Hz).

¹³C NMR (75.47 MHz, CDCl₃): δ 172.17, 161.02, 159.09, 137.57, 120.07,119.97, 57.69, 57.04, 52.35, 51.72, 51.54, 46.80, 46.29, 46.23.

The methyl ester of chloromethyl-2-pyridinecarboxylic acid was added toa solution of compound 4″ (0.939 g, 3.38 mmol) in acetonitrile (150 mL)in the presence of K₂CO₃ (1.8 g, 13.5 mmol). The mixture was stirred atroom temperature for one week and then filtered and concentrated. Thecrude product was purified by chromatography on a column of alumina(98/2 CH₂Cl₂/MeOH) to give compound 5″ in the form of a yellow oil(0.822 g, 42%).Hydrochloric acid (20 mL, 6M) was added slowly to compound 5″. Themixture was refluxed for 24 hours and then concentrated. The crudeproduct was purified using C18 HPLC (90/10 to 5/95 H₂O 0.1%HCl/acetonitrile) and the ligand 6″ was obtained in the form of acolorless oil (0.310 g, 35% calculated for 3 HCl).

¹H NMR (500 MHz, D₂O): δ 7.98-7.87 (m, 5H), 7.65 (d, 1H, ³J=7.3 Hz),7.47 (m, 1H), 7.43 (d, 1H, ³J=7.9 Hz), 7.31 (d, 1H, ³J=7.9 Hz), 4.78 (s,2H), 4.74 (s, br, 2H), 4.54 (s, 2H), 4.20 (s, 2H), 3.78 (s, br, 2H),3.63 (s, br, 2H), 3.55 (s, 2H), 3.12 (m, 4H).

¹³C NMR (125.77 MHz, D₂O): 172.25, 171.82, 170.66, 158.41, 153.72,153.30, 152.78, 152.14, 151.95, 143.56, 142.61, 142.36, 130.33, 129.50,127.63, 127.24, 125.33, 125.16, 61.91, 61.78, 61.72, 60.08, 56.17,56.12, 53.57, 53.37

7) Synthesis of the Ligand Pc2a1pa Asym P04216 of Formula (I) Via the“Oxalate” Route

The methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid (711 g,3.85 mmol) was added to a solution of compound 2″ (1.0 g, 3.85 mmol) inacetonitrile (300 mL) in the presence of K₂CO₃ (1.5 g, 12 mmol). Themixture was refluxed for 4 days and then filtered and concentrated. Thecrude product was purified by chromatography on a column of alumina(98/2 CH₂Cl₂/MeOH) to give compound 7″ in the form of a yellow oil (1.56g, 99%).Compound 7″ (1.56 g, 3.81 mmol) was dissolved in MeOH (40 mL) andconcentrated sulfuric acid (1 mL) was added slowly. The mixture wasrefluxed for 24 hours. After cooling to room temperature, the solventwas evaporated off. 20 mL of water were added and the pH was adjusted to7 with K₂CO₃. The water was evaporated off and the residue was taken upin dichloromethane. Magnesium sulfate was added and the organic phasewas filtered and then concentrated. The crude product was purified bychromatography on a column of alumina (98/2 to 95/5 CH₂Cl₂/MeOH) to give8″ in the form of a yellow oil (1.24 g, 92%).A solution of tert-butyl bromoacetate (1.36 g, 6.98 mmol) inacetonitrile (150 mL) was added to a solution of 8″ (1.24 g, 3.49 mmol)and K₂CO₃ (1.93 g, 14 mmol) in acetonitrile (150 mL). The mixture wasrefluxed for 2 days. The solvent was evaporated off and the residue wastaken up in dichloromethane, filtered and concentrated. Compound 9″ wasobtained in the form of a yellow oil and used in the following stepwithout further purification.Hydrochloric acid (20 mL, 6M) was added slowly to compound 9″. Themixture was refluxed for 24 hours and then concentrated. The crudeproduct was purified using C18 HPLC (90/10 to 5/95 H₂O 0.1%HCl/acetonitrile) and the ligand 10″ was obtained in the form of acolorless oil.

8) Synthesis of the Ligand Pc3pa P04221 of Formula (I):

The methyl ester of chloromethyl-2-pyridinecarboxylic acid (1.35 g, 7.28mmol) was added to a solution of compound 1″ (0.50 g, 2.43 mmol) inacetonitrile (350 mL) in the presence of K₂CO₃ (1 g, 7.28 mmol). Themixture was refluxed for two days and then filtered and concentrated.The crude product was purified by chromatography on a column of alumina(98/2 CH₂Cl₂/MeOH). Compound 11″ is obtained in the form of a yellow oil(862 mg, 54%).Hydrochloric acid (20 mL, 6M) was added to compound 11″ (862 mg, 1.32mmol). The mixture was refluxed for 48 hours and then concentrated. Thecrude product was purified by precipitation from acetone. Compound 12″was obtained in hydrochloride salt form (0.574 g, 57% calculated for 4HCl).

¹H NMR (300 MHz, D₂O): δ 7.5-7.25 (m, 8H), 7.12-7.09 (m, 2H), 6.75 (d,2H), 4.17 (s, 4H), 4.09 (s, 4H), 3.86 (s, 2H), 3.29 (m, 4H), 2.83 (m,4H).

¹³C NMR (75.47 MHz, D₂O): δ 170.11, 168.95, 158.31, 154.51, 153.79,150.12, 149.48, 145.95, 144.50, 143.67, 132.57, 132.24, 129.55, 126.76,62.60, 62.03, 60.78, 57.15, 54.14.

-   -   9-1) Synthesis of a Picolinate Bromide Derivative of Formula        (II)

Chelidamic acid monohydrate 1′″ (5 g, 24.9 mmol) and phosphoruspentabromide (34 g, 79.0 mmol) were heated at 90° C. Once a liquidmixture was obtained, heating was continued for 2 hours at 90° C. Aftercooling the mixture with ice, chloroform (100 mL) and MeOH (100 mL) wereadded. The solution is mixed for 20 hours at room temperature and the pHis adjusted to 7 with saturated NaHCO₃ solution. The solvents wereevaporated off and the aqueous phase was extracted using dichloromethane(3×100 mL). The organic phase was dried over MgSO₄, filtered andconcentrated to give compound 2′″ in the form of a white solid (6.43 g,94%).

¹H NMR (300 MHz, CDCl₃): δ 8.42 (s, 2H), 3.99 (s, 6H).

¹³C NMR (300 MHz, CDCl₃): δ 164.02, 149.12, 135.13, 131.33, 53.52.

Compound 2′″ (6.43 g, 23.5 mmol) was dissolved in dichloromethane (50mL) and methanol (70 mL). NaBH₄ (1.02 g, 28.2 mmol) was addedportionwise to the mixture at 0° C., under nitrogen. After stirring for4 hours, hydrochloric acid was added to adjust the pH to 5. The solventswere evaporated off and the pH of the aqueous phase was adjusted to 12with Na₂CO₃. The aqueous phase was extracted with dichloromethane (3×100mL) and the organic phase was dried with MgSO₄, filtered andconcentrated under vacuum. After purification on alumina, compound 3′″was obtained in the form of a white solid (3.92 g, 68%).

¹H NMR (300 MHz, CDCl₃): δ 8.12 (s, 1H), 7.76 (s, 1H), 4.82 (s, 2H),3.95 (s, 3H).

¹³C NMR (300 MHz, CDCl₃): δ 164.55, 162.33, 147.96, 134.66, 127.31,127.21, 64.49, 53.29.

Under an inert atmosphere, Pd(Ph₃)₂Cl₂ (232 mg, 0.33 mmol) and Cul(124.2, 0.65 mmol) were added to a degassed solution of 1-dodecyne (651mg, 3.92 mmol) in triethylamine (10 mL) and 3′″ (800 mg, 3.26 mmol) infreshly distilled THF. The mixture was stirred at 40° C. for 20 hours.After cooling to room temperature, the suspension was filtered andtriturated with Et₂O (40 mL). The filtrate was washed with saturatedNH₄Cl solution (2×50 mL) and brine (40 mL). Finally, the organic phasewas dried over MgSO₄, filtered and concentrated under vacuum. The crudeproduct was purified on silica gel (7/3 to 4/6 hexane/ethyl acetate) togive compound 4′″ in the form of a white solid (727 mg, 67%).

¹H NMR (300 MHz, CDCl₃): δ 7.93 (s, 1H), 7.48 (s, 1H), 4.80 (s, 2H),3.95 (s, 3H), 2.41 (t, 2H), 1.58 (m, 2H), 1.5-1.1 (m, 14H), 0.85 (t,3H).

¹³C NMR (300 MHz, CDCl₃): δ 165.37, 160.62, 147.05, 134.54, 126.15,125.98, 97.83, 78.05, 64.62, 53.02, 31.99, 29.67, 29.59, 29.40, 29.21,29.01, 28.39, 22.77, 19.60, 14.20.

Compound 4′″ (727 mg, 2.15 mmol) was dissolved in dichloromethane (80mL) and triethylamine (653 mg, 6.45 mmol). Mesyl chloride (369 mg, 3.23mmol) was added and the mixture was stirred at room temperature for 30minutes. The organic phase was washed with saturated aqueous sodiumhydrogen carbonate solution (100 mL) and then dried over MgSO₄, filteredand concentrated. Compound 5′″ is obtained in the form of a white solid(896 mg, quantitative yield).

-   -   9-2) Synthesis of the Ligand Pc1a2pa Asym C12 P04245 of Formula        (I):

A solution of compound 5′″ (712 mg, 1.75 mmol) in acetonitrile (50 mL)was added to a solution of compound 4″ (243 mg, 0.87 mmol) in refluxingacetonitrile (100 mL) in the presence of K₂CO₃ (361 g, 2.6 mmol). Themixture was refluxed for one week. After cooling to room temperature,the suspension was filtered and the solvent was evaporated off. Thecrude product was purified by chromatography on a column of alumina togive compound 7′″ in the form of a yellow oil.Production and Purification of the Ligand Pc1a2pa Asym C12 P04245:Saponification StepA solution of KOH (5 mL, 1M) was added to a solution of compound 7′″ (91mg, 0.10 mmol) in THF (6 mL). The mixture was stirred vigorously for 5hours at room temperature. The organic phase was evaporated and theresidue was then purified by exclusion chromatography (Sephadex LH20,100/0 to 90/10 CH₂Cl₂/MeOH). Product 8′″ was obtained in the form of acolorless solid (48 mg, 56%).

10) Synthesis of the Analog Pc1a2pa Asym C8 P04330:

-   -   10-1) Synthesis of a C8 Picolinate Bromide Derivative

Under an inert atmosphere, Pd(Ph₃)₂Cl₂ (246 mg, 0.35 mmol) and Cul (134,0.70 mmol) were added to a degassed solution of 1-octyne (464 mg, 4.21mmol) in triethylamine (10 mL) and 3′″ (863 mg, 3.51 mmol) in freshlydistilled THF (20 mL). The mixture was stirred at 40° C. for 20 hours.After cooling to room temperature, the suspension was filtered andtriturated with Et₂O (40 mL). The filtrate was washed with saturatedNH₄Cl solution (2×20 mL) and brine (20 mL). Finally, the organic phasewas dried over MgSO₄, filtered and concentrated under vacuum. The crudeproduct was purified on silica gel (7/3 to 4/6 hexane/ethyl acetate) togive compound 4″″ in the form of a white solid (573 mg, 59%).

¹H NMR (300 MHz, CDCl₃): δ 7.69 (s, 1H), 7.39 (s, 1H), 4.63 (s, 2H),3.74 (s, 3H), 2.22 (t, 2H), 1.40 (m, 2H), 1.24 (m, 2H), 1.35-1.15 (m,4H), 0.69 (t, 3H).

¹³C NMR (300 MHz, CDCl₃): δ 164.88, 161.27, 146.43, 133.96, 125.53,125.35, 97.05, 77.74, 64.26, 52.45, 30.96, 28.25, 27.94, 22.18, 19.12,13.66.

A solution of compound 4″″ (573 mg, 2.08 mmol) in anhydrous CH₂Cl₂ (50mL) was cooled to 0° C. under an inert atmosphere. PBr₃ (676 mg, 2.5mmol) was added and the mixture was then refluxed for 2 hours. Aftercooling to room temperature, the reaction medium was neutralized with 50mL of water and K₂CO₃ to pH 7. The organic phase was dried over MgSO₄,filtered and then concentrated under vacuum. After purification onsilica gel (9/1 to 4/6 hexane/ethyl acetate), product 5′″ was obtainedin the form of a white solid (289 mg, 41%).

¹H NMR (300 MHz, CDCl₃): δ 7.91 (s, 1H), 7.54 (s, 1H), 4.52 (s, 2H),3.93 (s, 3H), 2.37 (t, 2H), 1.54 (m, 2H), 1.37 (m, 2H), 1.3-1.2 (m, 4H),0.85 (t, 3H).

13C NMR (300 MHz, CDCl₃): δ 165.00, 157.39, 147.63, 134.85, 128.82,126.58, 98.23, 77.60, 53.04, 32.80, 31.25, 28.55, 28.18, 22.48, 19.48,14.01.

-   -   10-2) Synthesis of the Ligand Pc1a2pa Asym C8 P04330

Compound 5″″ (289 mg, 0.85 mmol) was added to a solution of compound 4″(106 mg, 0.38 mmol) in anhydrous acetonitrile (30 mL) in the presence ofK₂CO₃ (158 mg, 1.1 mmol). The mixture was refluxed for one week. Aftercooling to room temperature, the suspension was filtered and the solventwas evaporated off. The crude product was dissolved in a minimum amountof ethyl acetate and pentane was then added until the solution becamecloudy. The oil formed was rinsed with pentane and precipitated again.Compound 7″″ was obtained in the form of a yellow oil (155 mg, 51%).A solution of KOH (2 mL, 1M) was added to a solution of compound 7″″ (55mg, 0.069 mmol) in THF (5 mL). The mixture was stirred vigorously for 5hours at room temperature. The organic phase was evaporated and theresidue was then purified by exclusion chromatography (Sephadex LH20,100/0 to 90/10 CH₂Cl₂/MeOH). Product 8″″ was obtained in the form of acolorless solid (30 mg, 58%).

C— Study of the Compounds of Formula (I) and of the Complexes Accordingto the Invention

C-1 Synthesis of the Yttrium Complexes

1) The procedure for synthesizing the complex Y-Pc1a2pa sym P04183 isdescribed below and is applicable to all of the ligands of generalformula (I):

The ligand P04213 is dissolved in ultra-pure water and the pH isadjusted to 5 with 1M sodium hydroxide solution. The salt YCl₃.6H₂O (1.5eq) is dissolved in ultra-pure water. The yttrium solution is added tothe ligand solution with stirring. After adjusting the pH to 5, thesolution is refluxed overnight. The complex is then purified by HPLC onC18 (100/0 to 10/90 H₂O/ACN) according to the scheme below:

2) Synthesis of an yttrium-90 complex, P04233:

The ligand P04214 was used in a complexation reaction with yttrium-90 inorder to confirm the complexation results obtained with non-radioactivenatural yttrium. A radiolabeling study was performed.

The parameters studied are as follows:

Parameters Study range Optimum pH 1-9 6.5-9 Temperature 20-100° c. 80°c. Ligand concentration Mol/L 10⁻⁵-10⁻² mol/L 10⁻⁴-10⁻² mol/L Time Min.5-60 min. 15 min

In summary, the optimum conditions for labeling P04214 are yttrium-90 inacetate medium pH=6.5-9, the ligand P04214 between 10⁻⁴ and 10⁻² M inEtOH; 15 min at 80° C. The radiolabeling yield obtained is >90%(P04233).

3) Procedure for and result of labeling of the ligand P04245 withyttrium-90, production of the complex P04283:

The parameters studied are as follows:

Parameters Study range Optimum pH 4.65-9 6.5-9 Temperature 20-90° c. 50°c. Ligand concentration Mol/L 10⁻⁵-10⁻³ mol/L 10⁻³ mol/L Time Min. 5-60min. 15 minThe optimum labeling conditions are:

yttrium-90 in acetate medium;

pH=4.65-9;

ligand P04245 at 10⁻³ M in EtOH;

for 15 min at 50° C.

4) Procedure for and result of extraction of the complex P04283 withLipiodol, production of P04284:

The solution containing the complex P04283 was made up to 2 mL with 1 mLof saline, and an equivalent volume of Lipiodol (2 mL) was added to thesolution containing the complex. After stirring and centrifugation, thephases are separated and counted. The yield for extraction into Lipiodolis 89.8±5.0% (n=3).

5) Procedure for and results of the stability tests in humanphysiological saline:

Procedure for Preparing the Radiotracer P04284

1 mL of yttrium-90 acetate at pH=7 is added to 1 mL of ligand P04245dissolved in ethanol at a concentration of 10⁻³ mol/L to form thecomplex P04283. The solution is heated for 30 minutes at 90° C. 2 mL ofLipiodol are added and the mixture is stirred vigorously. The phases areseparated by centrifugation (3500 rpm, 15 minutes). The lipiodol-basedphase is collected and made up to 2 mL with Lipiodol to give theexpected radiotracer P04284.

1 mL of freshly prepared radiotracer is taken and then deposited in a 12mL flat-bottomed glass flask. The activity is measured with anactivimeter, and the time is noted. 10 mL of 0.9% saline solution(physiological saline) are added and the mixture is stirred. The flaskis then placed in the incubator set at 37° C., equipped with a stirrerset at 30 rpm (revolutions per minute).

The system is left stirring for several days. The aqueous phase issampled at various times to assay the yttrium-90 released. Each samplewas taken in triplicate.

The results are given in FIG. 5. The complexes formed according to theinvention and vectorized with Lipiodol are stable in physiologicalsaline.

C-2 Synthesis of the Lanthanide Complexes:

-   -   1) The complexation reactions of gadolinium with the ligands        P04218 and P04216 and also with ligand P04213 are performed in        water in the presence of one equivalent of GdCl₃ at pH 5-6 at        reflux overnight.

Example: Complexation of the Ligand P04216 with Gadolinium

Purification of the complexes is performed by preparative HPLC so as toremove the remaining salts.

-   -   2) Study of the Relaxivity of the Gadolinium Complexes:

The relaxivity studies were performed on the gadolinium complexes of theligands P04218, P04216 and P04213, on Minispec Mq-20 and Minispec Mq-60machines (Brüker, Karlsruhe, Germany) at 20 MHz (0.47 T) and 60 MHz (1.4T) in water at 37° C.

For each complex prepared previously, a [Gd]concentration rangeextending from 0.5 to 5 mM was prepared and the values T1 and T2 of eachof these solutions were then measured to determine the relaxivity valuesr1 and r2 by means of equation 1. For each of the ligands, a trend curvewhose correlation coefficient was equal to or very close to 1 wasobtained, which made it possible to check equation 1 and to validate thequality of the measurements taken. The curves plotted make it possibleto determine the relaxivity value “r” which corresponds to coefficient“a” of the equation for the straight line “ax+b”.

$r = {\frac{1}{\left\lbrack {Gd}^{3 +} \right\rbrack}\left( {\frac{1}{T_{obs}} - \frac{1}{T_{H\; 2O}}} \right)}$

Equation 1: General Formula for Calculating the Relaxivity Values r1 andr2

Relaxivity (mmol⁻¹s⁻¹) 20 MHz 60 MHz Pc2a1pa sym r1 = 3.9 r1 = 3.2P04218 r2 = 4.5 r2 = 3.9 Pc2a1pa asym r1 = 3.7 r1 = 3.2 P04216 r2 = 4.1r2 = 3.7 Pc1a2pa sym r1 = 1.9 r1 = 1.6 P04213 r2 = 2.1 r2 = 1.8

It is found that the relaxivities observed are of the same order ofmagnitude as those obtained with the gadolinium-based contrast agentsused clinically, for example Dotarem®.

-   -   3) Stability of the Gd Complexes in Competitive Medium:

To a solution comprising the gadolinium complex of ligands P04218 andP04216 at 2.5 mM in phosphate buffer at 333 mM is added a solution ofZnCl₂ at 2.5 mM. The relaxivity value of these solutions is measuredregularly. The relationship between the relaxivity measured at a giventime and that at t=0 min as a function of the time in the presence ofthe Zn solution is given in FIG. 6. The complexes according to theinvention are stable over time.

-   -   4) Synthesis and Characterization of the Complexes

General procedure for preparing the lanthanide complexes (Ln=Y³⁺, Gd³⁺,Eu³⁺, Tb³⁺, Yb³⁺, Lu³⁺).

The ligand is dissolved in water and the pH is adjusted to 5 with 1M KOHsolution and a solution of the metal chloride (M=Y³⁺, Gd³⁺, Eu³⁺, Tb³⁺,Yb³⁺, Lu³⁺) is then added (1.2 equivalents). The mixture is refluxedovernight and the solution obtained is concentrated.

The complex is purified by preparative chromatography on a column ofC-18 grafted silica, eluting with a water/acetonitrile mixture.

Abbreviation Ligand Mono S Pc-2a1pa Sym P04218 L1 Mono AS Pc-2a1pa AsymP04216 L3 Di Sym Pc-1a2pa sym P04213 L2 Di AS Pc-1a2pa Asym P04214 L4Synthesis of [ML1(H₂O)]

[YL1(H₂O)]L1.3HCl (27.2 mg, 0.048 mmol), YCl₃.6H₂O (25.0 mg, 0.082 mmol)Yield: 24.5 mg, 91%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅YN₅O₆]⁺, 544.0858; measured544.0858 [M+H]⁺, calc. [C₂₂H₂₆YN₅O₆]²⁺, 272.5465; measured 272.5469[M+2H]²⁺.[GdL1(H₂O)]L1.3HCl (36.5 mg, 0.064 mmol), GdCl₃.6H₂O (27.5 mg, 0.074 mmol)Yield: 39.8 mg, 98%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅GdN₅O₆]⁺, 613.1040; measured613.1031 [M+H]⁺, calc. [C₂₂H₂₆GdN₅O₆]²⁺, 307.0557; measured 307.0560[M+2H]²⁺.[EuL1(H₂O)]L1.3HCl (22.0 mg, 0.039 mmol), EuCl₃.6H₂O (17.1 mg, 0.047 mmol)Yield: 22.1 mg, 91%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅EuN₅O₆]⁺, 608.1012; measured608.1004 [M+H]⁺, calc. [C₂₂H₂₆EuN₅O₆]²⁺, 304.5542; measured 304.5544[M+2H]²⁺:[TbL1(H₂O)]L1.3HCl (22.0 mg, 0.039 mmol), TbCl₃.6H₂O (17.4 mg, 0.047 mmol)Yield: 22.6 mg, 95%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅TbN₅O₆]⁺, 614.1053; measured614.1048 [M+H]⁺, calc. [C₂₂H₂₆TbN₅O₆]²⁺, 307.5563; measured 307.5565[M+2H]²⁺.[YbL1(H₂O)]L1.3HCl (25.0 mg, 0.044 mmol), YbCl₃.6H2O (20.5 mg, 0.053 mmol)Yield: 27.3 mg, 96%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅YbN₅O₆], 629.1188; measured629.1187 [M+H]⁺, calc. [C₂₂H₂₆YbN₅O₆]²⁺, 315.0630; measured 315.0635[M+2H]²⁺.[LuL1(H₂O)]L1.3HCl (25.0 mg, 0.044 mmol), LuCl₃.6H₂O (20.6 mg, 0.053 mmol)Yield: 26 mg, 91%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂5LuN₅O₆]⁺, 630.1207; found630.1196 [M+H]⁺, calc. [C₂₂H₂₆LuN₅O₆]²⁺, 315.5640; found 315.5641[M+2H]²⁺.Synthesis of [ML2]

[YL2]L2.3HCl (100.0 mg, 0.155 mmol), YCl₃.6H2O (89.0 mg, 0.293 mmol)Yield: 84.8 mg, 88%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈YN₆O₆]⁺, 621.1123; measured621.1116 [M+H]⁺, calc. [C₂₇H₂₉YN₆O₆]²⁺, 311.0598; measured 311.0603[M+2H]²⁺.[GdL2]L2.3HCl (39.0 mg, 0.061 mmol), GdCl₃.6H2O (27.0 mg, 0.073 mmol)Yield: 41.1 mg, 99%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈GdN₆O₆]⁺, 690.1306; measured690.1313 [M+H]⁺, calcd. for [C₂₇H₂₉GdN₆O₆]²⁺, 345.5689; measured345.5690 [M+2H]²⁺.[EuL2]L2.3HCl (25.0 mg, 0.039 mmol), EuCl₃.6H₂O (17.1 mg, 0.047 mmol)Yield: 25.3 mg, 96%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈EuN₆O₆]⁺, 685.1277; measured685.1279 [M+H]⁺, calc. [C₂₇H₂₉EuN₆O₆]²⁺, 343.0675; measured 343.0680[M+2H]²⁺.[TbL2]L2.3HCl (20.0 mg, 0.031 mmol), TbCl₃.6H₂O (13.9 mg, 0.037 mmol)Yield: 19.6 mg, 92%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈TbN₆O₆]⁺, 691.1318; measured691.1314 [M+H]⁺, calc. [C₂₇H₂₉TbN₆O₆]^(2+,) 346.0696; measured 346.0697[M+2H]²⁺.[YbL2]L2.3HCl (22.0 mg, 0.034 mmol), YbCl₃.6H₂O (15.9 mg, 0.041 mmol)Yield: 22.1 mg, 92%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈YbN₆O₆]⁺, 706.1453; measured706.1454 [M+H]⁺, calc. [C₂₇H₂₉YbN₆O₆]^(2+,) 353.5763; measured 353.5764[M+2H]²⁺.[LuL2]L2.3HCl (22.0 mg, 0.034 mmol), LuCl₃.6H₂O (16.0 mg, 0.041 mmol)Yield: 22.8 mg, 95%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈LuN₆O₆]⁺, 707.1473; measured707.1476 [M+H]⁺, calc. [C₂₇H₂₉LuN₆O₆]²⁺, 354.0773; measured 354.0776[M+2H]²⁺.Synthesis of [ML3(H₂O)]

[YL3(H₂O)]L3.3HCl (30.0 mg, 0.053 mmol), YCl₃.6H₂O (24.0 mg, 0.079 mmol)Yield: 28.0 mg, 94%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅YN₅O₆]⁺, 544.0858; measured544.0859 [M+H]⁺ calc. [C₂₂H26YN₅O₆]^(2+,) 272.5465; measured 272.5469[M+2H]²⁺.[GdL3(H₂O)]L3.3HCl (53.0 mg, 0.093 mmol), GdCl₃.6H2O (41.3 mg, 0.111 mmol)Yield: 58.7 mg, 99%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅GdN₅O₆]⁺, 613.1040; measured613.1030 [M+H]⁺, calc. [C₂₂H₂₆GdN₅O₆]²⁺, 307.0557; measured 307.0568[M+2H]²⁺.[EuL3(H₂O)]L3.3HCl (28.5 mg, 0.050 mmol), EuCl₃.6H₂O (22.1 mg, 0.060 mmol)Yield: 29.0 mg, 92%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅EuN₅O₆]⁺, 608.1012; measured608.1007 [M+H]⁺, calc. [C₂₂H₂₆EuN₅O₆]^(2+,) 304.5542; measured 304.5544[M+2H]²⁺.[TbL3(H₂O)]L3.3HCl (24.0 mg, 0.042 mmol), TbCl₃.6H2O (19.0 mg, 0.051 mmol)Yield: 23.2 mg, 89%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅TbN₅O₆]⁺, 614.1053; measured614.1049 [M+H]⁺, calc. [C₂₂H₂₆TbN₅O₆]^(2+,) 307.5563; measured 307.5563[M+2H]²⁺.[YbL3(H₂O)]L3.3HCl (25.0 mg, 0.044 mmol), YbCl₃.6H2O (20.5 mg, 0.053 mmol)Yield: 27.8 mg, 98%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅YbN₅O₆]⁺, 629.1188; measured629.1182 [M+H]⁺, calc. [C₂₂H₂₆YbN₅O₆]^(2+,) 315.0630; measured 315.0634[M+2H]²⁺.[LuL3(H₂O)]L3.3HCl (28.0 mg, 0.049 mmol), LuCl₃.6H₂O (23.1 mg, 0.059 mmol)Yield: 29 mg, 91%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₂H₂₅LuN₅O₆]⁺, 630.1207; measured630.1204 [M+H]⁺, calc. [C22H26LuN5O6]²⁺, 315.5640; measured 315.5642[M+2H]²⁺.Synthesis of [ML4(H₂O)]

[YL4]L4.3HCl (30.0 mg, 0.047 mmol), YCl₃.6H₂O (24.0 mg, 0.079 mmol)Yield: 24.8 mg, 92%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈YN₆O₆]⁺, 621.1123; measured621.1121 [M+H]⁺, calc. [C₂₇H₂₉YN₆O₆]²⁺, 311.0598; measured 311.0601[M+2H]²⁺.[GdL4]L4.3HCl (36.2 mg, 0.056 mmol), GdCl₃.6H₂O (25.1 mg, 0.068 mmol)Yield: 38.1 mg, 98%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈GdN₆O₆]⁺, 690.1306; measured690.1321 [M+H]⁺, calc. [C₂₇H₂₉GdN₆O₆]²⁺, 345.5698; measured 345.5690[M+2H]²⁺.[EuL4]L4.3HCl (23.5 mg, 0.036 mmol), EuCl₃.6H₂O (16.0 mg, 0.044 mmol)Yield: 21.8 mg, 87%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈EuN₆O₆]⁺, 685.1277; measured685.1277 [M+H]⁺, calc. [C₂₇H₂₉EuN₆O₆]²⁺, 343.0675; measured 343.0680[M+2H]²⁺.[TbL4]L4.3HCl (24.0 mg, 0.037 mmol), TbCl₃.6H₂O (16.4 mg, 0.044 mmol)Yield: 25.3 mg, 98%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈TbN₆O₆]^(+,) 691.1318;measured 691.1316 [M+H]⁺ calc. [C₂₇H₂₉TbN₆O₆]²+, 346.0696; measured346.0699 [M+2H]²⁺.[YbL4]L4.3HCl (30.0 mg, 0.047 mmol), YbCl₃.6H₂O (21.7 mg, 0.056 mmol)Yield: 30.4 mg, 93%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈YbN₆O₆]⁺, 706.1453; measured706.1454 [M+H]⁺, calc. [C₂₇H₂₉YbN₆O₆]²⁺, 353.5763; measured 353.5768[M+2H]²⁺.[LuL4]L4.3HCl (30.0 mg, 0.047 mmol), LuCl₃.6H₂O (21.8 mg, 0.056 mmol)Yield: 30.4 mg, 92%ESI-HR-MS (positive, H₂O) m/z calc. [C₂₇H₂₈LuN₆O₆]⁺, 707.1473; measured707.1470 [M+H]⁺, calc. [C₂₇H₂₉LuN₆O₆]^(2+,) 354.0773; measured 354.0776[M+2H]²⁺.C-3 Study in Solution

1) Study by Nuclear Magnetic Resonance:

By way of example, the ¹H NMR spectra of the ligand P04213 and of itsyttrium complex P04183 recorded in D₂O are shown in FIG. 1. Relative tothe spectrum of the ligand, the presence of the metal cation generatesdissymmetry and thus a larger number of signals (cf. FIG. 1).

2) Study by UV-Visible Spectroscopy:

The absorption spectra of the ligands and of their yttrium complexeswere recorded in water at pH 3.8 and 5.5 (acetate buffer). Theabsorption band corresponding to the π-π* transitions of pyridine extendfrom 240 to 300 nm for the ligands and the complexes (cf. FIG. 2).

C-4 Complexation Kinetics

The complexation kinetics of the ligands Pc1a2pa sym P04213, Pc1a2paasym P04214 and Pc2a1pa sym P04218 with yttrium were studied at pH 3.8and pH 5.5 in acetate buffer medium by UV-visible spectroscopy. Placedat the absorption maximum of the complex, the increase in absorbanceintensity is measured every two seconds until the maximum absorbance isreached. The decrease in intensity of the absorbance at the absorbancemaximum of the ligand is monitored when the absorption band of thecomplex is masked by that of the ligand. For this study, theconcentration of the ligands Pc1a2pa sym and Pc1a2pa asym is 4×10⁻⁵ Mand 8×10⁻⁵ M for the ligand Pc2a1pa sym. At pH 5.5 and 3.8, the ligandPc1a2pa sym has the fastest complexation kinetics, with totalcomplexation in 30 and 400 seconds, respectively. For the ligandsPc1a2pa asym and Pc2a1pa sym, the complexation is complete in 1100seconds at pH 3.8 and 100 seconds at pH 5.5.

Complexation is thus rapid for all of the ligands under the conditionsstudied. (cf FIG. 3).

C-5 Kinetic Inertia in Competitive Medium

The dissociation kinetics of the complexes in concentrated acidic mediummakes it possible to determine the behavior of the complexes in highlycompetitive medium. The rate of decomplexation is monitored byUV-visible spectroscopy, with C_(YL)=4×10⁻⁵ M for the complexesY-Pc1a2pa sym P04183, Y-Pc1a2pa asym P04215 and Y-Pc2a1pa sym P04219, in0.5, 1, 2, 4 and 5M HCl medium. The absorption band of the complexdisappears more or less rapidly to reveal the absorption band of theligand at shorter wavelengths. The plot of the increase in intensity ofthe absorbance at the absorption maximum of the ligand as a function oftime (A=f(t)) makes it possible to determine the half-life timest_(1/2). The t_(1/2) values for the various complexes are listed in thetable below. The complexes may be classified in the following mannerfrom the most inert to the least inert: Y-Pc1a2pa asym>>Y-Pc1a2pasym>Y-PCTA>Y-PCTMB>Y-Pc2a1pa sym. The presence of two picolinate arms onthe pyclene macrocycle increases the inertia of the yttrium complex inacidic medium. Furthermore, the inertia of the complex Y-Pc1a2pa asym isgreater than that of its symmetrical analog, with, respectively, at_(1/2) of 433 minutes in 5 M HCl medium, as opposed to 8.5 minutes.

Ligands PCTMB Pc1a2pa sym Pc1a2pa asym Pc2a1pa sym PCTA C_(HCl) t_(1/2)(min) 0.5M   37 347 >1 week 55 95 (in progress) 1M 20 140 (in progress)27 39 2M 9 51 2745 10.6 17 4M 3.2 13 907 2.7 6.7 5M 2.6 8.5 433 0.8 3.1C-6 Studies of Thermodynamic Stability by Potentiometry

1) Protonation Constants of the Liqands

Four protonation constants were determined for the ligands Pc1a2pa symP04213, Pc1a2pa asym P04214, Pc2a1pa sym P04216 and Pc3pa P04221. Thesevalues are coherent with those determined for PCTMB (phosphonic acid,P,P′,P″-[3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triyltris(methylene)]tris-,P, P′, P″-tributyl ester) and also with those described in theliterature especially for PCTA(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triaceticacid), EDTA (ethylenediaminetetraacetic acid) and DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

TABLE 13 Ligands PCTMB EDTA ² PCTA ³ DOTA ⁴ Pc1a2pa sym Pc1a2pa asymPc2a1pa sym I 0.1 KNO₃ 0.1 KNO₃ 1.0 KCl 0.1 Me₄NNO₃ 0.1 KNO₃ 0.1 KNO₃0.1 KNO₃ log K_(t) ^(H) [HL]/[L][H] 11.16 10.22 11.36 12.09 11.30 10.5010.43 [H₂L]/[HL][H] 5.28 6.16 7.35 9.76 5.58 6.73 7.38 [H₃L]/[H₂L][H]1.72 2.71 3.83 4.56 4.23 3.86 3.95 [H₄L]/[H₃L][H] — 2.0 2.12 4.09 3.012.98 2.15 [H₅L]/[H₄L][H] — — 1.29 — — — — Σlog K_(i) 18.15 21.09 25.9530.50 24.12 24.06 23.90For the derivative Pc3pa P04221, the values obtained are as follows:

TABLE 14 Pc3pa P04221 logK^(H) _(i) [HL]/[L][H] 10.03 [H₂L]/[HL][H] 5.95[H₃L]/[H₂L][H] 3.82 [H₄L]/[H₃L][H] 2.98

2) Stability Constants of the Complexes

The thermodynamic protonation and stability constants of the complexeswere determined by potentiometry at 25° C. with control of the ionicstrength (I=0.1 M KNO₃). Refinement of the titration curves with theHyperQuad software makes it possible to determine the overall constants(log β), from which the partial constants (log K) are calculated.

The stability constants of the ligands Pc1a2pa sym P04213, Pc1a2pa asymP04214 et Pc2a1pa sym P04218 and P04221 with yttrium were determined bydirect potentiometric titration. The log K_(YL) constant values for theligands Pc1a2pa sym, Pc1a2pa asym and Pc2a1pa sym are, respectively,19.78, 19.49 and 19.28, and the log K_(YLH) ⁻¹ constant values are11.84, 11.79 and 10.60.

Reaction equilibrium PCTMB ^(b) EDTA ^(c) PCTA ^(f) DOTA ^(g) Pc1a2pasym Pc1a2pa asym Pc2a1pa sym Log K_(MHiL) Y³⁺ [ML]/[M][L] 19.49 18.5^(d) 20.28 24.9 ^(h) 19.78 19.49 19.28 [MHL]/[ML][H] 3.45 — 1.81 — — — —[ML]/[MLOH][H] 9.10 — 11.10 — 11.84 11.79 10.60 ^(a) For the sake ofclaritiy, the charges are not indicated. ^(b) Values determined bycompetition with EDTA. 0.1M KNO₃. ^(c) Ref 2, 0.1M KNO₃. ^(d) Ref ⁵,0.1M NMe₄Cl. ^(e) Ref ⁶, 0.1M KNO₃. ^(f) Ref 3, 1.0M KCl. ^(g) Ref 4,0.1M NMe₄Cl. ^(h) Ref ⁷, 0.1M NMe₄NO₃.

TABLE 16 Pc3pa + Y³⁺P04222 logK_(MHiL) ML]/[M][L] 16.42 [MHL]/[ML][H]3.11 [ML]/[MLOH][H] 11.02These stability constants are not comparable as such: the basicity ofthe ligands needs to be taken into account. The constant pM=−log[M] isused for this purpose. It is calculated from the protonation constantsof the ligands and the stability constants of the complexes withCL=10×CM=10⁻⁵ M at pH 7.4. The ligand Pc1a2pa asym P04214 has a p(Y) of17.3, which is higher than that of PCTA (p(Y)=17.0), of Pc1a2pa symP04213 (p(Y)=16.8) and of Pc2a1pa sym P04218 (p(Y)=16.9). The highestp(Y) nevertheless remains that of DOTA, with a value of 18.8.

TABLE 17 pM = −log[M] ^(a) Ligands PCTMB EDTA PCTA DOTA Pc1a2pa symPc1a2pa asym Pc2a1pa sym pY 16.7 16.6 17.0 18.8 16.8 17.3 16.9 ^(a)Values calculated from the constants of the preceding tables. with C_(L)= 10 × C_(M) = 10⁻⁵ M à pH 7.4.For Pc3pa P04222, the pM calculated is 14.7.The speciation diagrams, plotted from the thermodynamic stabilityconstants of the yttrium complexes, indicate that the complexes existexclusively in the form YL over a wide pH range, including at pH 7.4.

REFERENCES

-   1 Aime, S.; Botta, M.; Geninatti Crich, S.; Giovenzana, G. B.;    Jommi, G.; Pagliarin, R.; Sisti, M. Inorg. Chem.    1997, 36, 2992-3000.-   2 Delgado, R.; Figueira, C.; Quintino, S. Talanta 1997, 45, 451.-   3 Tircsó, G.; Kovacs, Z.; Dean Sherry, A. Inorg. Chem. 2006, 45,    9269.-   4 Chaves, S.; Delgado, R.; Frausto da Silva, J. J. R. Talanta 1992,    39, 249.-   5 Kumar, K.; Chang C. A.; Francesconi, L. C.; Dischino, D. D.;    Malley, M. F.; Gougoutas, J. Z.; Tweedle,    M. F. Inorg. Chem. 1994, 33, 3567.-   6 Delgado, R.; Frausto da Silva, J. J. R. Talanta 1982, 29, 815.-   7 Cox, J. P. L.; Jankowski, K. J.; Kataky, R.; Parker, D.;    Beeley, N. R. A.; Boyce, B. A.; Eaton, M. A. W.; Millar,    K.; Millican, A. T.; Harrison, A.; Walkerc, C. J. Chem. Soc. Chem.    Commun. 1989, 797.    C-7 Solid-State Study

The yttrium complex P04183 crystallizes in water. The structure obtainedby x-ray diffraction is presented below. The metal is coordinated to thefour nitrogen atoms of the macrocycle, the two nitrogen atoms of thepicolinate arms and the three oxygen atoms of the carboxylic acids. Thecoordination sphere of the metal is N6O3, i.e. 9 coordinant atoms. Thehelicities Δ and Λ derived from the orientation of the picolinate andacetate arms are both present: the complex thus crystallizes as aracemic mixture.

The invention claimed is:
 1. A compound of general formula (X)

in which: R₁and R₂ represent, independently of each other, one of thefollowing: H, a (C₁-C₂₀)alkyl group or a (C₁-C₂₀)alkylene-(C₆-C₁₀)arylgroup, wherein said alkyl, alkylene and aryl groups optionally aresubstituted with one or more organic acid functions; X₁, X₂ and X₃represent, independently of each other, one of the following: H,—C(O)N(Re)(Rd), a (C₁-C₂₀)alkyl, a (C₂-C₂₀)alkenyl, a (C₂-C₂₀)alkynyland a (C₆-C₁₀)aryl, with Re and Rd being, independently of each other, Hor a (C₁-C₂₀) alkyl group, wherein said alkyl, alkenyl and alkynylgroups optionally comprise one or more heteroatoms and/or one or more(C₆-C₁₀)arylenes in their chains, and wherein said alkyl, alkenyl andalkynyl groups optionally are substituted with a (C₆-C₁₀)aryl; whereinsaid alkyl, alkenyl, alkynyl and aryl groups optionally are substitutedwith one or more organic acid functions; and Y₁ represents a C(O)OHgroup, a protected form of the C(O)OH group in which the hydrogen isreplaced with an alkyl, or a group of formula (II)

in which the radicals Ri represent, independently of each other, one ofthe following: H, halogen, N₃, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl and (C₆-C₁₀)aryl, wherein said alkyl, alkenyl andalkynyl groups optionally comprise one or more heteroatoms and/or one ormore (C₆-C₁₀)arylenes in their chains and, wherein said alkyl, akenyl,and alkynyl groups optionally are substituted with a (C₆-C₁₀) aryl;wherein said alkyl, alkenyl, alkynyl and aryl groups optionally aresubstituted with one or more organic acid functions.
 2. The compound ofclaim 1, wherein X₁, X₂ and X₃ represent, independently of each other,one of the following: H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl and(C₂-C₂₀)alkynyl, wherein said alkyl, alkenyl and alkynyl groupsoptionally comprise one or more heteroatoms in their chain.
 3. Thecompound of claim 2, wherein X₁, X₂ and X₃ are H.
 4. The compound ofclaim 1, wherein the organic acid functions optionally substituting thealkyl, alkenyl, alkynyl and aryl groups of the X₁, X₂ and X₃ are,independently of each other, one of the following: —COOH, —SO₂OH,—P(O)(OH)₂ and —O—P(O)(OH)₂.
 5. The compound of claim 1, wherein R₁ andR₂ represent, independently of each other, H or (C₁-C₂₀)alkyl group. 6.The compound of claim 5, wherein R₁ and R₂ represent H.
 7. The compoundof claim 1, wherein the organic acid functions optionally substitutingthe alkyl, alkylene and aryl groups of R₁ and R₂ represent,independently of each other, one of the following: —COOH, —SO₂OH,—P(O)(OH)₂ and —O—P(O)(OH)₂.
 8. The compound of claim 1, wherein theradicals Ri represent, independently of each other, one of thefollowing: H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl and (C₂-C₂₀)alkynyl,wherein said alkyl, alkenyl and alkynyl groups optionally comprise oneor more N, O and S heteroatoms.
 9. The compound of claim 1, wherein theradicals Ri represent, independently of each other, H or(C₂-C₁₅)alkynyl.
 10. The compound of claim 1, wherein the organic acidfunctions substituting the alkyl, alkenyl, alkynyl and aryl groups ofthe radicals Ri represent, independently of each other, one of thefollowing: —COOH, —SO₂OH, —P(O)(OH)₂ and —O—P(O)(OH)₂.
 11. The compoundof claim 10, wherein the organic acid functions substituting the alkyl,alkenyl, alkynyl and aryl groups of the radicals Ri represent —COOH. 12.The compound of claim 1, wherein the group of formula (II) is thefollowing group:


13. The compound of claim 1, wherein: R₁ and R₂ represent H; and Y₁represents C(O)OH or the following formula:


14. A method of preparing the compound of claim 1, the methodcomprising: functionalizing a compound of general formula (IX)

by adding to the nitrogen atom of the NH group thereof a —C(R₁)(R₂)—Y₁group wherein: R₁ and R₂ represent, independently of each other, one ofthe following: H, a (C₁-C₂₀)alkyl group or a(C₁-C₂₀)alkylene-(C₆-C₁₀)aryl group, wherein said alkyl, alkylene andaryl groups optionally are substituted with one or more organic acidfunctions; Y₁ represents a C(O)OH group, a protected form of the C(O)OHgroup in which the hydrogen is replaced with an alkyl, or a group offormula (II)

in which the radicals Ri represent, independently of each other, one ofthe following: H, halogen, N₃, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl and (C₆-C₁₀)aryl, wherein said alkyl, alkenyl andalkynyl groups optionally comprise one or more heteroatoms and/or one ormore (C₆-C₁₀)arylenes in their chains and, wherein said alkyl, akenyl,and alkynyl groups optionally are substituted with a (C₆-C₁₀)aryl;wherein said alkyl, alkenyl, alkynyl and aryl groups optionally aresubstituted with one or more organic acid functions; to form thecompound (X) of claim
 1. 15. The compound of claim 1, wherein the alkylthat replaced the hydrogen of the protected form of the C(O)OH group ofY₁ is selected from the group consisting of tert-butyl and methyl.